Amino ceramide-like compounds and therapeutic methods of use

ABSTRACT

The present invention provides amino ceramide-like compounds which inhibit glucosyl ceramide (GlyCer) formation by inhibiting the enzyme GlyCer synthase, thereby lowering the level of glycosphingolipids. The compounds of the present invention have improved GlcCer synthase inhibition activity and are therefore useful in therapeutic methods for treating various conditions and diseases associated with altered glycosphingolipid levels.

RELATED APPLICATIONS

This application is a continuation of U.S. Application Ser. No.10/839,497, filed May 5, 2004 now U.S. Pat. No. 6,916,802, which is acontinuation of U.S. application Ser. No. 10/134,315, filed Apr. 29,2002, now abandoned. The entire teachings of these prior applicationsare incorporated herein by reference.

SPONSORSHIP

The present invention was supported by grant nos. R01 DK41487, R01DK69255 and RO139255 from the National Institutes of Health, contractR43 CA 58159 from the National Cancer Institute, grant GM 35712 from theNational Institute of General Medical Sciences, and by the University ofMichigan Comprehensive Cancer Center grant 2P30 CA 46592 from theNational Cancer Institute, U.S. Public Health Service, DHHS. Grantnumber for Merit Award from Veteran's Administration. The government mayhave certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to ceramide-like compounds and,more particularly, to ceramide-like compounds containing a tertiaryamine group and their use in therapeutic methods.

BACKGROUND OF THE INVENTION

Hundreds of glycosphingolipids (GSLs) are derived from glucosylceramide(GlcCer), which is enzymatically formed from ceramide and UDP-glucose.The enzyme involved in GlcCer formation is UDP-glucose:N-acylsphingosineglucosyltransferase (GlcCer synthase). The rate of GlcCer formationunder physiological conditions may depend on the tissue level ofUDP-glucose, which in turn depends on the level of glucose in aparticular tissue (Zador, I. Z. et al., “A Role for GlycosphingolipidAccumulation in the Renal Hypertrophy of Streptozotocin-Induced DiabetesMellitus,” J. Clin. Invest., 91:797-803 (1993)). In vitro assays basedon endogenous ceramide yield lower synthetic rates than mixturescontaining added ceramide, suggesting that tissue levels of ceramide arealso normally rate-limiting (Brenkert, A. et al., “Synthesis ofGalactosyl Ceramide and Glucosyl Ceramide by Rat Brain: Assay Proceduresand Changes with Age,” Brain Res., 36:183-193 (1972)).

It has been found that the level of GSLs controls a variety of cellfunctions, such as growth, differentiation, adhesion between cells orbetween cells and matrix proteins, binding of microorganisms and virusesto cells, and metastasis of tumor cells. In addition, the GlcCerprecursor, ceramide, may cause differentiation or inhibition of cellgrowth (Bielawska, A. et al., “Modulation of Cell Growth andDifferentiation by Ceramide,” FEBS Letters, 307:211-214 (1992)) and beinvolved in the functioning of vitamin D₃, tumor necrosis factor-α,interleukins, and apoptosis (programmed cell death). The sphingols(sphingoid bases), precursors of ceramide, and products of ceramidecatabolism, have also been shown to influence many cell systems,possibly by inhibiting protein kinase C (PKC).

It is likely that all the GSLs undergo catabolic hydrolysis, so anyblockage in the GlcCer synthase should ultimately lead to depletion ofthe GSLs and profound changes in the functioning of a cell or organism.An inhibitor of GlcCer synthase, PDMP(1R-phenyl-2R-decanoylamino-3-morpholino-1-propanol), previouslyidentified as the D-threo isomer (Inokuchi, J. et al., “Preparation ofthe Active Isomer of 1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol,Inhibitor of Glucocerebroside Synthetase,” J. Lipid Res., 28:565-571(1987)), has been found to produce a variety of chemical andphysiological changes in cells and animals (Radin, N. S. et al., “Use of1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol (PDMP), an Inhibitor ofGlucosylceramide Synthesis,” In NeuroProtocols, A Companion to Methodsin Neurosciences, S. K. Fisher et al., Ed., (Academic Press, San Diego)3:145-155 (1993) and Radin, N. S. et al., “Metabolic Effects ofInhibiting Glucosylceramide Synthesis with PDMP and Other Substances,”In Advances in Lipid Research; Sphingolipids in Signaling, Part B., R.M. Bell et al., Ed. (Academic Press, San Diego) 28:183-213 (1993)).Particularly interesting is the compound's ability to cure mice ofcancer induced by Ehrlich ascites carcinoma cells (Inokuchi, J. et al.,“Antitumor Activity in Mice of an Inhibitor of GlycosphingolipidBiosynthesis,” Cancer Lett., 38:23-30 (1987)), to produce accumulationof sphingosine and N,N-dimethylsphingosine (Felding-Habermann, B. etal., “A Ceramide Analog Inhibits T Cell Proliferative Response ThroughInhibition of Glycosphingolipid Synthesis and Enhancement ofN,N-Dimethylsphingosine Synthesis,” Biochemistry, 29:6314-6322 (1990)),and to slow cell growth (Shayman, J. A. et al., “Modulation of RenalEpithelial Cell Growth by Glucosylceramide: Association with ProteinKinase C, Sphingosine, and Diacylglyceride,” J. Biol. Chem.266:22968-22974 (1991)). Compounds with longer chain fatty acyl groupshave been found to be substantially more effective (Abe, A. et al.,“Improved Inhibitors of Glucosylceramide Synthesis,” J. Biochem.,111:191-196 (1992)).

The importance of GSL metabolism is underscored by the seriousness ofdisorders resulting from defects in GSL metabolizing enzymes (whichdiseases may collectively be referred to as “glycosphingolipidoses”).For example, Tay-Sachs, Gaucher's, and Fabry's diseases, resulting fromenzymatic defects in the GSL degradative pathway and the accumulation ofGSL in the patient, all have severe clinical manifestations. Anotherexample of the importance of GSL function is seen in a mechanism bywhich blood cells, whose surfaces contain selectins, can, under certainconditions, bind to GSLs in the blood vessel walls and produce acute,life-threatening inflammation (Alon, R. et al., “Glycolipid Ligands forSelectins Support Leukocyte Tethering & Rolling Under Physiologic FlowConditions,” J. Immunol., 154:5356-5366 (1995)).

At present there is only one treatment available for patients withGaucher disease, wherein the normal enzyme which has been isolated fromnormal human tissues or cultured cells is administered to the patient.As with any drug isolated from human material, great care is needed toprevent contamination with a virus or other dangerous substances.Treatment for an individual patient is extremely expensive, costinghundreds of thousands, or even millions of dollars, over a patient'slifetime. It would thus be desirable to provide a treatment whichincludes administration of a compound that is readily available and/orproducible from common materials by simple reactions.

Possibly of even greater clinical relevance is the role of glucolipidsin cancer. For example, it has been found that certain GSLs occur onlyin tumors; certain GSLs occur at abnormally high concentrations intumors; certain GSLs, added to tumor cells in culture media, exertmarked stimulatory or inhibitory actions on tumor growth; antibodies tocertain GSLs inhibit the growth of tumors; the GSLs that are shed bytumors into the surrounding extracellular fluid inhibit the body'snormal immunodefense system; the composition of a tumor's GSLs changesas the tumors become increasingly malignant; and, in certain kinds ofcancer, the level of a GSL circulating in the blood gives usefulinformation regarding the patient's response to treatment. Because ofthe significant impact GSLs have on several biochemical processes, thereremains a need for compounds having improved GlcCer synthase inhibitionactivity.

It would thus be desirable to provide compounds which inhibit GlcCersynthase activity, thereby lowering the level of GSLs and increasing GSLprecursor levels, e.g. increasing the levels of ceramide and sphingols.It would further be desirable to provide compounds which inhibit GlcCersynthase activity and lower the level of GSLs without also increasingceramide levels. It would also be desirable to provide compounds andtherapeutic methods to treat conditions and diseases associated withaltered GSL levels and/or GSL precursor levels.

SUMMARY OF THE INVENTION

Novel compounds are provided which inhibit GlcCer formation byinhibiting the enzyme GlcCer synthase, thereby lowering the level ofGSLs. The compounds of the present invention have improved GlcCersynthase inhibition activity and are, therefore, highly useful intherapeutic methods for treating various conditions and diseasesassociated with altered GSL levels, as well as GSL precursor levels. Forexample, the compounds of the present invention may be useful in methodsinvolving cancer growth and metastasis, the growth of normal tissues,the ability of pathogenic microorganisms to bind to normal cells, thebinding between similar cells, the binding of toxins to human cells, andthe ability of cancer cells to block the normal process of immunologicalcytotoxic attack.

Additional objects, advantages, and features of the present inventionwill become apparent from the following description and appended claims,taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent toone skilled in the art by reading the following specification andsubjoined claims and by referencing the following drawings in which:

FIG. 1 is a graph showing the growth and survival of 9 L gliosarcomacells grown in medium containing different GlcCer synthase inhibitors;

FIG. 2 is a graph showing the protein content of MDCK cells cultured for24 hr in medium containing different concentrations of the separatederythro- and threo-isomers of a preferred compound of the presentinvention;

FIG. 3 is a graph showing [³H]thymidine incorporation into the DNA ofMDCK cells treated with a preferred compound of the present invention;

FIGS. 4A and 4B are graphs showing the effects of P4 and p-methoxy-P4 onGlcCer synthase activity;

FIG. 5 is a graph showing the linear relationship between the inhibitionof GlcCer synthase activity and electronic parameter (δ) and hydrophobicparameter (π);

FIG. 6 is a graph showing the effects of dioxy P4 derivatives on GlcCersynthase activity;

FIG. 7 is a bar graph showing the effects of D-t-3′,4′-ethylenedioxy-P4on GlcCer synthesis and cell growth;

FIG. 8 is a schematic of the synthetic pathway for4′-hydroxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol;

FIG. 9 is an illustration of the structures of P4 and ofphenyl-substituted P4 homologues;

FIG. 10 is an HPLC chromatogram showing the separation of theenantiomers of P4 and p-methoxy-P4 by chiral chromatography;

FIG. 11 is a graph showing the effects of D-threo-4′-hydroxy-P4 ascompared to D-threo-p-methoxy-P4 on GlcCer synthase activity;

FIG. 12 is a graph showing the effects of D-threo enantiomers ofP4,4′-hydroxy-P4 and 3′,4′-ethylenedioxy-P4 on 1-O-acyceramide synthaseactivity;

FIG. 13 is a graph showing the effect of D-threo-P4 on GlcCer synthesisand cell growth;

FIG. 14 is a graph showing the effect of D-threo-4′-hydroxy-P4 on GlcCersynthesis and cell growth; and

FIG. 15 is a graph showing the effect of D-threo-3′,4′-ethylenedioxy-P4on GlcCer synthesis and cell growth.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Novel compounds are provided which inhibit GlcCer formation byinhibiting the enzyme GlcCer synthase, thereby lowering the level ofGSLs. The compounds of the present invention have improved GlcCersynthase inhibitory activity and are, therefore, highly useful intherapeutic methods for treating various conditions and diseasesassociated with altered GSL levels.

The compounds of the present invention generally have the followingformula:

wherein

R¹ is a phenyl group, preferably a substituted phenyl group such asp-methoxy, hydroxy, dioxane substitutions such as methylenedioxy,ethylenedioxy, and trimethylenedioxy, cyclohexyl or other acyclic group,t-butyl or other branched aliphatic group, or a long alkyl or alkenylchain, preferably 7 to 15 carbons long with a double bond next to thekernel of the structure. The aliphatic chain can have a hydroxyl groupnear the two asymmetric centers, corresponding to phytosphingosine.

R² is an alkyl residue of a fatty acid, 2 to 18 carbons long. The fattyacid can be saturated or unsaturated, or possess a small substitution atthe C-2 position (e.g., a hydroxyl group). It is contemplated that theR² group fatty acid may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, or 18 carbons long. Longer fatty acids also may be useful.Preferrably R² in the above structure is either 5 carbons or 7 carbonsin length.

R³ is a tertiary amine, preferably a cyclic amine such as pyrrolidine,azetidine, morpholine or piperidine, in which the nitrogen atom isattached to the kernel (i.e., a tertiary amine).

All four structural isomers of the compounds are contemplated within thepresent invention and may be used either singly or in combination (i.e.,DL-threo or DL-erythro).

The preferred aliphatic compound of the present invention isD-threo-1-pyrrolidino-1-deoxyceramide, identified as IV-231B herein andalso referred to as PD. The preferred aromatic compound of the presentinvention is 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol,identified as BML-119 herein and also referred to as P4. The structuresof the preferred compounds are as follows:

Additional preferred compounds of the present invention areD-t-3′,4′-ethylenedioxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol,also referred to herein as D-t-3′,4′-ethylenedioxy-P4, andD-t-4′-hydroxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol, alsoreferred to herein as D-t-4′-hydroxy-P4.

By increasing the acyl chain length of PDMP from 10 to 16 carbon atoms,the efficacy of the compounds of the present invention as GlcCersynthase inhibitors is greatly enhanced. The use of a less polar cyclicamine, especially a pyrrolidine instead of a morpholine ring, alsoincreases the efficacy of the compounds. In addition, replacement of thephenyl ring by a chain corresponding to sphingosine yields a stronglyinhibitory material. By using a chiral synthetic route, it wasdiscovered that the isomers active against GlcCer synthase had theR,R-(D-threo)-configuration. However, strong inhibition of the growth ofhuman cancer cells in plastico was produced by both the threo anderythro racemic compounds, showing involvement of an additional factorbeyond simple depletion of cell glycosphingolipids by blockage of GlcCersynthesis. The growth arresting effects could be correlated withincreases in cellular ceramide and diglyceride levels.

Surprisingly, the aliphatic pyrrolidino compound of the presentinvention (identified as IV-231B), was strongly inhibitory toward theGlcCer synthase and produced almost complete depletion of glycolipids,but did not inhibit growth or cause an accumulation of ceramide.Attempts were made to determine if the differences in growth effectscould be attributed to the influence of the inhibitors on relatedenzymes (ceramide and sphingomyelin synthase and ceramidase andsphingomyelinase). While some stimulation or inhibition of enzymeactivity was noted, particularly at high inhibitor concentrations (50μM), these findings did not explain the differing effects of thedifferent inhibitors.

By slowing the synthesis of GlcCer, the compounds of the presentinvention lower the levels of all the GlcCer-derived GSLs due to the GSLhydrolases which normally destroy them. While the body will continue tomake the more complex GSLs from available GlcCer, the rate of synthesiswill slow down as the level of GlcCer diminishes. The rate of loweringdepends on the normal rate of destruction of each GSL. These rates,however, are relatively rapid in animals and cultured cells.

At higher dosages, many of the compounds of the present inventionproduce an elevation in the level of ceramide. Presumably this occursbecause cells continue to make ceramide despite their inability toutilize it for GlcCer synthesis. Ceramide is also normally converted tosphingomyelin, but this process does not seem to be able to handle theexcess ceramide. It has been unexpectedly found, however, that anadditional process is also involved, since even those isomers that areinert against GlcCer synthase also produce an elevation in ceramidelevels. Moreover, the blockage of GlcCer synthase can occur at lowinhibitor dosages, yet ceramide accumulation is not produced. Thepreferred aliphatic compound of the present invention,D-threo-1-pyrrolidino-1-deoxyceramide (PD), does not produce ceramideaccumulation at all, despite almost complete blockage of GlcCersynthesis.

This distinction between the aromatic and the aliphatic compounds of thepresent invention is important because ceramide has recently beenproposed to cause cell death (apoptosis) by some still unknownmechanism. At lower dose levels, the aromatic compounds of the presentinvention cause GSL disappearance with only small accumulation ofceramide and inhibition of cell growth. Higher dosages cause much moreceramide deposition and very slow cell growth or cell death.

In certain embodiments, the inventors found that compounds containing a16 carbon fatty acyl group is an extremely efficient and potent GlcCersynthase inhibitor. However, the longer the acyl chain of the PDMP-basedcompounds, the more lipophilic the agent. The inventors found that theC16 fatty acyl PDMP derivatives had a long retention time within thebody. In some instances, it may be desirable to produce compounds havinga C6 or C8 fatty acyl chain (i.e., R² in the above structures is a C5 orC7 fatty acyl chain backbone). Specifically contemplated by the presentinvention are compounds of the following formulas:

In one embodiment of the present invention, methods of treating patientssuffering from inborn genetic errors in the metabolism of GlcCer and itsnormal anabolic products (lactosylceramide and the more complex GSLs)are provided. The presently known disorders in this category includeGaucher, Fabry, Tay-Sachs, Sandhoff, and GM1 gangliosidosis. The geneticerrors lie in the patient's inability to synthesize a hydrolytic enzymehaving normal efficiency. Their inefficient hydrolase allows the GSL togradually accumulate to a toxic degree, debilitating or killing thevictim. The compounds of the present invention slow the formation ofGSLs, thus allowing the defective hydrolase to gradually “catch up” andrestore the concentrations of GSLs to their normal levels and thus thecompounds may be administered to treat such patients.

With respect to Gaucher disease, it has been calculated that much of thepatient's accumulated GlcCer in liver and spleen arises from the bloodcells, which are ultimately destroyed in these organs after they havereached the end of their life span. The actual fraction, lipid derivedfrom blood cells versus lipid formed in the liver and spleen cells, isactually quite uncertain, but the external source must be important.Therefore, it is necessary for the compounds of the present invention todeplete the blood cells as they are formed or (in the case of whiteblood cells) while they still circulate in the blood. Judging fromtoxicity tests, the white cells continue to function adequately despitetheir loss of GSLs. Although the toxicity studies were not of a longenough duration to produce many new red cells with low GSL content, itis possible that circulating red cells also undergo turnover (continualloss plus replacement) of GSLs.

In an alternative embodiment of the present invention, for the treatmentof disorders involving cell growth and division, high dosages of thecompounds of the present invention are administered but only for arelatively short time. These disorders include cancer, collagen vasculardiseases, atherosclerosis, and the renal hypertrophy of diabeticpatients. Accumulation or changes in the cellular levels of GSLs havebeen implicated in these disorders and blocking GSL biosynthesis wouldallow the normal restorative mechanisms of the body to resolve theimbalance.

With atherosclerosis, it has been shown that arterial epithelial cellsgrow faster in the presence of a GlcCer product (lactosylceramide).Oxidized serum lipoprotein, a material that normally circulates in theblood, stimulates the formation of plaques and lactosylceramide in theinner lining of blood vessels. Treatment with the compounds of thepresent invention would inhibit this mitogenic effect.

In an additional embodiment of the present invention, patients sufferingfrom infections may be treated with the compounds of the presentinvention. Many types of pathogenic bacteria have to bind to specificGSLs before they can induce their toxic effects. As shown in Svensson,M. et al., “Epithelial Glucosphingolipid Expression as a Determinant ofBacterial Adherence and Cytokine Production,” Infect. and Immun.,62:4404-4410 (1994), expressly incorporated by reference, PDMP treatmentreduces the adherence of E. coli to mammalian cells. Several viruses,such as influenza type A, also must bind to a GSL. Several bacterialtoxins, such as the verotoxins, cannot themselves act without firstbinding to a GSL. Thus, by lowering the level of GSLs, the degree ofinfection may be ameliorated. In addition, when a patient is alreadyinfected to a recognizable, diagnosable degree, the compounds of thepresent invention may slow the further development of the infection byeliminating the binding sites that remain free.

It has been shown that tumors produce substances, namely gangliosides, afamily of GSLs, that prevent the host i.e., patient, from generatingantibodies against the tumor. By blocking the tumor's ability to secretethese substances, antibodies against the tumor can be produced. Thus, byadministering the GlcCer synthase inhibitors of the present invention tothe patient, the tumors will become depleted of their GSLs and thebody's normal immunological defenses will come into action and destroythe tumor. This technique was described in Inokuchi, J. et al.,“Antitumor Activity in Mice of an Inhibitor of GlycosphingolipidBiosynthesis,” Cancer Lett., 38:23-30 (1987), expressly incorporated byreference. The compounds of the present invention and in particular thealiphatic compounds require much lower doses than those previouslydescribed. This is particularly important because the lower dose mayreduce certain side effects. Moreover, because the aliphatic compoundsof the present invention do not produce ceramide accumulation, they areless toxic. In addition,1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), may act via twopathways, GSL depletion and ceramide accumulation.

In an alternative embodiment, a vaccine-like preparation is provided.Here, cancer cells are removed from the patient (preferably ascompletely as possible), and the cells are grown in culture in order toobtain a large number of the cancer cells. The cells are then exposed tothe inhibitor for a time sufficient to deplete the cells of their GSLs(generally 1 to 5 days) and are reinjected into the patient. Thesereinjected cells act like antigens and are destroyed by the patient'simmunodefense system. The remaining cancer cells (which could not bephysically removed) will also be attacked by the patient's antibodies.In a preferred embodiment, the patient's circulating gangliosides in theplasma are removed by plasmapheresis, since the circulating gangliosideswould tend to block the immunodefense system.

It is believed that tumors are particularly dependent on GSL synthesisfor maintenance of their growth (Hakomori, S. “New Directions in CancerTherapy Based on Aberrant Expression of Glycosphingolipids:Anti-adhesion and Ortho-Signaling Therapy,” Cancer Cells 3:461-470(1991)). Accumulation of ceramide in treated tumors also slows theirgrowth or kills them. Tumors also generate large amounts of GSLs andsecrete them into the patient's body, thereby preventing the host'snormal response by immunoprotective cells, which should generateantibodies against or otherwise destroy tumor cells (e.g., tumors areweakly antigenic). It has also been shown that GSL depletion blocks themetastasis of tumor cells (Inokuchi, J. et al., “Inhibition ofExperimental Metastasis of Murine Lewis Long Carcinoma by an Inhibitorof Glucosylceramide Synthase and its Possible Mechanism of Action,”Cancer Res., 50:6731-6737 (1990). Tumor angiogenesis (e.g., theproduction of blood capillaries) is strongly influenced by GSLs (Ziche,M. et al., “Angiogenesis Can Be Stimulated or Repressed in In Vivo by aChange in GM3:GD3 Ganglioside Ratio,” Lab. Invest., 67:711-715 (1992)).Depleting the tumor of its GSLs should block the tumors from generatingthe new blood vessels they need for growth.

A further important characteristic of the compounds of the presentinvention is their unique ability to block the growth of multidrugresistant (“MDR”) tumor cells even at much lower dosages. This wasdemonstrated with PDMP by Rosenwald, A. G. et al., “Effects of theGlycosphingolipid Synthesis Inhibitor, PDMP, on Lysosomes in CulturedCells,” J. Lipid Res., 35:1232 (1994), expressly incorporated byreference. Tumor cells that survive an initial series of therapeutictreatments often reappear some years later with new properties—they arenow resistant to a second treatment schedule, even with different drugs.This change has been attributed to the appearance in the tumor of largeamounts of a specific MDR protein (P-glycoprotein). It has beensuggested that protein kinase C (PKC) may be involved in the action orformation of P-glycoprotein (Blobe, G. C. et al., “Regulation of PKC andIts Role in Cancer Biology,” Cancer Metastasis Rev., 13:411-431 (1994)).However, decreases in PKC have other important effects, particularlyslowing of growth. It is known that PDMP does lower the cellular contentof PKC (Shayman, J. A. et al., “Modulation of Renal Epithelial CellGrowth by Glucosylceramide: Association with Protein Kinase C,Sphingosine, and Diacylglyceride,” J. Biol. Chem., 266:22968-22974(1991)) but it is not clear why it so effectively blocks growth of MDRcells (Rosenwald, A. G. et al., “Effects of the GlycosphingolipidSynthesis Inhibitor, PDMP, On Lysosomes in Cultured Cells,” J. LipidRes., 35:1232 (1994)). A recent report showed that several lipoidalamines that block MDR action also lower the level of the enzyme acidsphingomyelinase (Jaffrezou, J. et al., “Inhibition of Lysosomal AcidSphingomyelinase by Agents which Reverse Multidrug Resistance,” Biochim.Biophys. Acta, 1266:1-8 (1995)). One of these agents was also found toincrease the cellular content of sphingosine 5-fold, an effect seen withPDMP as well. One agent, chlorpromazine, behaves like the compounds ofthe present invention, in its ability to lower tissue levels of GlcCer(Hospattankar, A. V. et al., “Changes in Liver Lipids AfterAdministration of 2-Decanoylamino-3-Morpholinopropiophenone andChlorpromazine,” Lipids, 17:538-543 (1982)).

It will be appreciated by those skilled in the art that the compounds ofthe present invention can be employed in a wide variety ofpharmaceutical forms; the compound can be employed neat or admixed witha pharmaceutically acceptable carrier or other excipients or additives.Generally speaking, the compound will be administered orally orintravenously. It will be appreciated that therapeutically acceptablesalts of the compounds of the present invention may also be employed.The selection of dosage, rate/frequency and means of administration iswell within the skill of the artisan and may be left to the judgment ofthe treating physician or attending veterinarian. The method of thepresent invention may be employed alone or in conjunction with othertherapeutic regimens. It will also be appreciated that the compounds ofthe present invention are also useful as a research tool e.g. to furtherinvestigate GSL metabolism.

The following Specific Example further describes the compounds andmethods of the present invention.

SPECIFIC EXAMPLE 1

The following formulas set forth preferred aromatic and aliphaticcompounds:

identified as (1R,2R)-1-phenyl-2-acylamino-3-cyclic amino-1-propanol,and referred to herein as the “aromatic inhibitors,” wherein

The phenyl group can be a substituted phenyl group (such asp-methoxyphenyl).

R′ is an alkyl residue of a fatty acid, 2 to 18 carbons long. The fattyacid can be saturated or unsaturated, or possess a small substitution atthe C-2 position (e.g., a hydroxyl group). It is contemplated that theR′ group fatty acid may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, or 18 carbons long. Longer fatty acids also may be useful.Preferrably R′ in the above structure is either 5 carbons or 7 carbonsin length.

R is morpholino, pyrrolidino, piperidino, azetidino (trimethyleneimino),N-methylethanolamino, diethylamino or N-phenylpiperazino. A smallsubstituent, such as a hydroxyl group, is preferably included on thecyclic amine moiety.

identified as (2R,3R)-2-palmitoyl-sphingosyl amine or 1-cyclicamino-1-deoxyceramide or 1-cyclicamino-2-hexadecanoylamino-3-hydroxy-octadec-4,5-ene, and referred toherein as the “aliphatic inhibitors,” wherein

R′ is an alkyl residue of a fatty acid, 2 to 18 carbons long. The fattyacid can be saturated or unsaturated, or possess a small substitution atthe C-2 position (e.g., a hydroxyl group). It is contemplated that theR′ group fatty acid may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, or 18 carbons long. Longer fatty acids also may be useful.Preferrably R′ in the above structure is either 5 carbons or 7 carbonsin length.

R is morpholino, pyrrolidino, piperidino, azetidino (trimethyleneimino),N-methylethanolamino, diethylamino or N-phenylpiperazino. A smallsubstituent, such as a hydroxyl group, is preferably included on thecyclic amine moiety.

The long alkyl chain shown in Formula II can be 8 to 18 carbon atomslong, with or without a double bond near the asymmetric carbon atom(carbon 3). Hydroxyl groups can, with advantage, be substituted alongthe aliphatic chain, particularly on carbon 4 (as in the naturallyoccurring sphingol, phytosphingosine). The long chain can also bereplaced by other aliphatic groups, such at t-butyl or cyclopentyl.

The aromatic inhibitors (see Formula I and Table 1) were synthesized bythe Mannich reaction from 2-N-acylaminoacetophenone, paraformaldehyde,and a secondary amine as previously described (Inokuchi, J. et al.,“Preparation of the Active Isomer of1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol, Inhibitor ofGlucocerebroside Synthetase,” J. Lipid Res., 28:565-571 (1987) andVunnam, R. R. et al., “Analogs of Ceramide that Inhibit GlucocerebrosideSynthetase in Mouse Brain,” Chem. Phys. Lipids, 26:265-278 (1980)). Forthose syntheses in which phenyl-substituted starting materials wereused, the methyl group in the acetophenone structure was brominated andconverted to the primary amine. Bromination of p-methoxyacetophenone wasperformed in methanol. The acetophenones and amines were from AldrichChemical Co., St. Louis, Mo. Miscellaneous reagents were from SigmaChemical Co. and the sphingolipids used as substrates or standards wereprepared by methods known in the art. The reactions produce a mixture offour isomers, due to the presence of two asymmetric centers.

The aliphatic inhibitors (See Formula II and Table 2) were synthesizedfrom the corresponding 3-t-butyldimethylsilyl-protected sphingols,prepared by enantioselective aldol condensation (Evans, D. A. et al.,“Stereoselective Aldol Condensations Via Boron Enolates,” J. Am. Chem.Soc., 103:3099-3111 (1981) and Abdel-Magid, A. et al., “Metal-AssistedAldol Condensation of Chiral A-Halogenated Imide Enolates: AStereocontrolled Chiral Epoxide Synthesis,” J. Am. Chem. Soc.,108:4595-4602 (1986)) using a modification of the procedure of Nicolaouet al. (Nicolaou, K. C. et al., “A Practical and EnantioselectiveSynthesis of Glycosphingolipids and Related Compounds. Total Synthesisof Globotriaosylceramide (Gb₃),” J. Am. Chem. Soc., 110:7910-7912(1988)). Each protected sphingol was first converted to thecorresponding primary triflate ester, then reacted with a cyclic amine.Subsequent N-acylation and desilylation led to the final products ingood overall yield (Carson, K. G. et al., “Studies onMorpholinosphingolipids: Potent Inhibitors of GlucosylceramideSynthase,” Tetrahedron Lett., 35:2659-2662 (1994)). The compounds can becalled 1-morpholino-(or pyrrolidino)-1-deoxyceramides.

Labeled ceramide, decanoyl sphingosine, was prepared by reaction of theacid chloride and sphingosine (Kopaczyk, K. C. et al., “In VivoConversions of Cerebroside and Ceramide in Rat Brain,” J. Lipid Res.,6:140-145 (1965)) and NBD-SM(12-[N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)]-sphingosylphosphorylcholine)was from Molecular Probes, Inc., Eugene, Oreg.

Methods

TLC of the amines was carried out with HPTLC plates (E. Merck silica gel60) and C-M-HOAc 90:10:10 (solvent A) or 85:15:10 (solvent B) orC-M-conc. ammonium hydroxide 30:10:1 (solvent C). The bands were stainedwith iodine or with Coomassie Brilliant Blue R-250 (Nakamura, K. et al.,“Coomassie Brilliant Blue Staining of Lipids on Thin-Layer Plates,”Anal. Biochem., 142:406-41 (1984)) and, in the latter case, quantifiedwith a Bio-Rad Model 620 videodensitometer operated with reflected whitelight. The faster band of each PDMP analog, previously identified as theerythro form, corresponds to the 1S,2R and 1R,2S stereoisomers, and theslower band, previously identified as the threo form, corresponds to the1R,2R and 1S,2S stereoisomers.

TLC of the cell lipids was run with C-M-W 24:7:1 (solvent D) or 60:35:8(solvent E).

Growth of cell lines. Comparisons of different inhibitors with regard tosuppression of human cancer cell growth were made by the University ofMichigan Cancer Center in vitro Drug Evaluation Core Laboratory. MCF-7breast carcinoma cells, HT-29 colon adenocarcinoma cells, H-460 lunglarge cell carcinoma cells, and 9 L brain gliosarcoma cells were grownin RPMI 1640 medium with 5% fetal bovine serum, 2 mM glutamine, 50units/ml of penicillin, 50 mg/ml of streptomycin, and 0.1 mg/ml ofneomycin. UMSCC-10A head and neck squamous carcinoma cells were grown inminimal essential medium with Earle salts and the same supplements.Medium components were from Sigma Chemical Co. Cells were plated in96-well microtiter plates (1000 cells/well for H-460 and 9 L cells, and2000 cells/well for the other lines), and the test compounds were added1 day later. The stock inhibitor solutions, 2 mM in 2 mM BSA, werediluted with different amounts of additional 2 mM BSA, then eachsolution was diluted 500-fold with growth medium to obtain the finalconcentrations indicated in the Figures and Tables.

Five days after plating the H-460 and 9 L cells, or 6 days for the otherlines, cell growth was evaluated by staining the adhering cells withsulforhodamine B and measuring the absorbance at 520 nm (Skehan, P. etal., “New Colorimetric Cytotoxicity Assay for Anticancer-DrugScreening,” J. Natl. Cancer Inst., 82:1107-1112 (1990)). The absorbanceof the treated cultures is reported as percent of that of controlcultures, to provide an estimate of the fraction of the cells thatsurvived, or of inhibition of growth rate.

For the experiments with labeled thymidine, each 8.5 cm dish contained500,000 Madin-Darby canine kidney (MDCK) cells in 8 ml of Dulbeccomodified essential supplemented medium. The cells were incubated at 37°C. in 5% CO₂ for 24 h, then incubated another 24 h with mediumcontaining the inhibitor-BSA complex. The control cells were alsoincubated in the presence of BSA. The cells were washed withphosphate/saline and trichloroacetic acid, then scraped off the dishes,dissolved in alkali, and analyzed for protein and DNA incorporatedtritium. [Methyl-³H]thymidine (10 μCi) was added 4 h prior toharvesting.

Assay of sphingolipid enzymes. The inhibitors were evaluated for theireffectiveness against the GlcCer synthase of MDCK cell homogenates byincubation in a thermostatted ultrasonic bath (Radin N. S. et al.,“Ultrasonic Baths as Substitutes for Shaking Incubator Baths,” Enzyme,45:67-70 (1991)) with octanoyl sphingosine anduridinediphospho[³H]glucose (Shukla, G. S. et al., “GlucosylceramideSynthase of Mouse Kidney: Further Characterization and Improved AssayMethod,” Arch. Biochem. Biophys., 283:372-378 (1990)). The lipoidalsubstrate (85 μg) was added in liposomes made from 0.57 mgdioleoylphosphatidylcholine and 0.1 mg of Na sulfatide. Confluent cellswere washed, then homogenized with a micro-tip sonicator at 0° C. for3×30 sec; ˜0.2 mg of protein was used in each assay tube. In the case ofthe aromatic inhibitors, the test compound was simply evaporated todryness from solution in the incubation tube. This method of adding theinhibitor was found to give the same results as addition as a part ofthe substrate liposomes. The aliphatic inhibitors, which appeared to beless soluble in water, were added as part of the substrate liposomes.

Acid and neutral ceramidases were assayed under conditions like thoseabove, but the medium contained 110 μM [1-¹⁴C]decanoyl sphingosine (10⁵cpm) in 340 μM dioleoylphosphatidylcholine liposomes and 0.34 mg of MDCKcellular protein homogenate. The acid enzyme was incubated in 32.5 mMcitrate-Na⁺ (pH 4.5) and the neutral enzyme buffer was 40 mM Tris-Cl⁻(pH 7.1 at 37° C.). After 60 min in the ultrasonic bath, 3 ml of C-M2:1, carrier decanoic acid, and 0.6 ml of 0.9% saline were added and thelipids in the lower layer were separated by TLC with C-HOAc 9:1. Theliberated decanoic acid was scraped off the glass plate and counted.

Ceramide synthase was assayed with 1 μM [3-³H]sphingosine (70,000 cpm,repurified by column chromatography), 0.2 mM stearoyl-CoA, 0.5 mMdithiothreitol, and ˜300 μg of MDCK homogenate protein in 25 mMphosphate-K⁺ buffer, pH 7.4, in a total volume of 0.2 ml. The incubation(for 30 min) and TLC were carried out as above and the ceramide band wascounted.

Sphingomyelin synthase was evaluated with 44 μM [¹⁴C]decanoylsphingosine (10⁵ cpm) dispersed with 136 μM dioleoyllecithin as in theceramide synthase assay, and 5 mM EDTA and 50 mM Hepes-Na⁺ pH 7.5, in atotal volume of 0.5 ml. MDCK homogenate was centrifuged at 600×gbriefly, then at 100,000×g for 1 h, and the pellet was suspended inwater and sonicated with a dipping probe. A portion of this suspensioncontaining 300 μg of protein was used. Incubation was at 37° C. for 30min, after which the lipids were treated as above, using C-M-W 60:35:8for the isolation of the labeled decanoyl SM.

Acid and neutral SMase assays were based on the procedures of Gatt etal. (Gatt, S. et al., “Assay of Enzymes of Lipid Metabolism With Coloredand Fluorescent Derivatives of Natural Lipids,” Meth. Enzymol.,72:351-375 (1981)), using liposomes containing NBD-SM dispersed like thelabeled ceramide (10 μM substrate and 30 μM lecithin). The assay mediumfor the neutral enzyme also contained 50 mM Tris-Cl⁻ (pH 7.4), 25 mMKCl, 5 mM MgCl₂ and 0.29 mg of MDCK cell protein in a total volume of0.25 ml. Incubation was at 37° C. for 30 min in the ultrasonic bath,then the fluorescent product, NBD-ceramide, was isolated by partitioningthe assay mixture with 0.45 ml 2-propanol, 1.5 ml heptane, and 0.2 mlwater. After centrifugation, a trace of contaminating NBD-SM was removedfrom 0.9 ml of the upper layer by washing with 0.35 ml water. The upperlayer was analyzed with a fluorometer (460 nm excitation, 515 nmemission).

Acid SMase was assayed with the same liposomes in 0.2 ml of assaymixture containing 125 mM NaOAc (pH 5.0) and 61 μg of cell protein, with60 min of incubation at 37° C. The resultant ceramide was determined asabove.

Results

Table 1 lists the aromatic compounds (see Formula I) synthesized andtheir migration rates on silica gel TLC plates. Separation of the threo-and erythro-steroisomers by TLC was generally very good, except forBML-120, -121, and -122 in the acidic solvent. In the basic solventBML-119 and BML-122 yielded poorly resolved double bands. BML-112 wasunexpectedly fast-running, especially when compared with BML-120; bothare presumably dihydrochlorides.

TABLE 1 STRUCTURES OF THE AROMATIC INHIBITORS BML Number Phenyl TLC hR₁or Name R Group Substituent Value^(a) PDMP^(b) morpholino 34(47) PPMPmorpholino (53) 112 N-phenylpiperazino 56 113 morpholino p-fluoro 25 114diethylamino 25 115 piperidino 29 (pentamethyleneimino) 116hexamethyleneimino 34 117^(b) morpholino p-fluoro 41 118 piperidinop-fluoro 26 119 pyrrolidino 20–70(44) (tetramethyleneimino) 1201-methylpiperazino 7–62 121 3- 1–30 dimethylaminopiperidino 122N-methylethanolamino 6–71 123 azetidino 12 (trimethyleneimino) 124 amino15 125 morpholino p-methoxy 37 126 pyrrolidino p-methoxy (50) ^(a)Onlythe relative R_(f) value of the faster-moving band is shown. The firstvalue was obtained with solvent A, the second with solvent C, and thenumbers in parentheses, with solvent B. In the case of BML-117, −125,and −126, a 20-cm high TLC plate was used to improve the separation.^(b)The fatty acid chain suggested by the R′ group is decanoyl, notpalmitoyl.

Table 2 describes four aliphatic inhibitors (see Formula II), which canbe considered to be ceramide analogs in which the C-1 hydroxyl group isreplaced by a cyclic amine. It should be noted that the carbonframeworks of compounds in Tables 1 and 2 are numbered differently (seeFormulas I and II), thus affecting comparisons of stereochemicalconfigurations. The threo- and erythro-isomers separated very poorly onTLC plates. Like the aromatic inhibitors, however, the morpholinecompounds ran faster than the pyrrolidine compounds. The latter arepresumably more strongly adsorbed by the silica gel because they aremore basic.

TABLE 2 CHARACTERIZATION OF THE SPHINGOSYL INHIBITORS Sphingol Number RGroup Structure TLC hR_(f) Value^(a) IV-181A morpholino 2R, 3S 43IV-206A morpholino 2R, 3R 40 IV-230A pyrrolidino 2R, 3S 31 IV-231Bpyrrolidino 2R, 3R 31 ^(a)TLC solvent: C-M-HOAc 90:5:10. Similar butfaster migrations were obtained with solvent A.

Structure-activity correlations. The results of testing the compounds inan assay system for GlcCer synthase are listed in Table 3. Eachinhibition determination (±SD) shown in Table 3 was carried out intriplicate. Some of the inhibitors were tested as mixtures ofDL-erythro- and DL-threo-isomers (see column 4). Only the D-threoenantiomer in each mixture was predicted to be the actual enzymeinhibitor (Inokuchi, J. et al., “Preparation of the Active Isomer of1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol, Inhibitor ofGlucocerebroside Synthetase,” J. Lipid Res., 28:565-571 (1987)); thecontent of this isomer was calculated by measuring the proportions ofthe threo- and erythro-racemic mixtures by quantitative TLC. TheDL-threo contents were found to be in the range of 40 to 72%. Thecomparisons, in the case of the mixtures, are therefore approximate(most of the samples were not purified to remove the three less-activeisomers and the observed data were not corrected for the level of theprimary enantiomers). The separation of the threo- and erythro-forms ismost conveniently accomplished by crystallization, but the specificconditions vary for each substance; thus only BML-119, a stronginhibitor, was separated into its threo- and erythro-forms. BML-112 isnot included in Table 3 because it had no inhibitory activity againstGlcCer synthase of rabbit liver microsomes.

TABLE 3 Inhibition of Ceramide Glucosyltransferase of MDCK cellHomogenates by Different Compounds % Inhibition at Inhibitor Number 80μM Inhibition at 5 μM Active Isomer^(h) BML-113 60 ± 4.7^(a) 29 BML-11431 ± 2.9^(a) 20 BML-115 84 ± 0.8^(a) 12.4 ± 0.7^(f) 27 82 ± 0.3^(b)BML-116 28 ± 3.2^(a) 27 BML-117 35 ± 0.6^(b) 36 BML-118 62 ± 0.4^(b) 8.3 ± 1.4^(f) 32 BML-119 94 ± 1.4^(b)   51 ± 2.3^(e) 29 97 ± 0.1^(c)  49 ± 0.8^(f) 96 ± 0.1^(d) BML-120 11 ± 3.0^(c) 26 BML-121 11 ± 0.4^(c)28 BML-122 58 ± 1.6^(d) 26 BML-123 86 ± 0.1^(d)   15 ± 0.8^(f) 33BML-124 −2 ± 1.6^(d) 15 BML-125   9 ± 3.0^(e) 26 BML-126 60 ± 1.8^(e)  54 ± 0.3^(f) 34 PDMP 90 ± 0.8^(a)   16 ± 1.8^(f) 100 PPMP   32 ±1.8^(e) 100   32 ± 0.7^(f) IV-181A   12 ± 0.2^(g) 100 IV-206A   73 ±1.5^(g) 100 IV-230A   19 ± 2.1^(g) 100 IV-231B   87 ± 0.4^(g) 100^(a–g)Different samples were assayed as parts of different experiments.^(h)Percent of the active D-stereoisomer in the synthesized sample,estimated by scanning the two stained bands, assuming the slower one wasthe (racemic) active form.

Comparison of PDMP (1R,2R-decanoate) and PPMP (1R,2R-palmitate), whenevaluated at the same time in Expt. f, shows that an increase in thechain length of the N-acyl group from 10 to 16 carbon atoms distinctlyimproved the inhibitory activity against GlcCer synthase, as notedbefore (Abe, A. et al., “Improved Inhibitors of GlucosylceramideSynthesis,” J. Biochem., 111:191-196 (1992)). Accordingly, most of theother compounds were synthesized with the palmitoyl group for comparisonwith PPMP. The comparisons between the best inhibitors are clearer atthe 5 μM level.

Replacing the oxygen in the morpholine ring of PPMP with a methylenegroup (BML-115) improved activity ˜1.4-fold (calculated from theinhibitions at 5 μM in Expt. f and relative purities, and assuming thatthe percent inhibition is proportional to concentration in this region:12.4/27×100/32=1.4). Previous comparison with mouse brain, humanplacenta, and human Gaucher spleen glucosyltransferase also showed thatreplacing the morpholino ring with the piperidino ring in a ketoneanalog of PDMP (1-phenyl-2-decanoylamino-3-piperidino-1-propanone)produced a much more active inhibitor (Vunnam, R. R. et al., “Analogs ofCeramide that Inhibit Glucocerebroside Synthetase in Mouse Brain,” Chem.Phys. Lipids, 26:265-278 (1980)).

Replacing the piperidine group with a 7-membered ring (BML-116) greatlydecreased the activity, while use of a 5-membered ring (BML-119)quadrupled the effectiveness (50 vs 12.4% inhibition). A 4-membered ring(BML-123) yielded a compound about as effective as the piperidinocompound. The parent amine (BML-124), its N,N-diethyl analog (BML-114),and the sterically bulky N-phenylpiperazine analog (BML-112) displayedlittle or no activity.

Replacing a hydrogen atom with a fluorine atom in the p-position of thephenyl ring decreased the inhibitory power (BML-117 vs PDMP and BML-118vs BML-115). Substitution of the p-position with an electron-donatingmoiety, the methoxy group, had a similar weakening effect in the case ofthe morpholino compound (BML-125 vs PPMP). Comparison of the pyrrolidinocompounds, which are more basic than the morpholino compounds, showedthat the methoxy group enhanced the inhibitory power (BML-126 vsBML-119).

Preparations of BML-119 were separated into threo and erythro racemicmixtures by HPLC on a Waters Microbondapak C₁₈ column, using M-W-conc.NH₄OH 90:10:0.2 as the elution solvent. The material eluting earlier(but migrating more slowly on a TLC plate) was called BML-130; the latereluting material (faster by TLC) was called BML-129. Assay of GlcCersynthase with each preparation at 5 μM showed 15% inhibition by BML-129and 79% inhibition by BML-130. TLC analysis of the two preparationsrevealed incomplete separation, which could explain the minor inhibitionby BML-129. When the two stereoisomers were separated by preparativeTLC, the difference in effectiveness was found to be somewhat higher,evidently due to the better separation by this method. Thus, theslower-migrating stereoisomer accounted for all or nearly all of theinhibitory activity, as noted with PDMP (Inokuchi, J. et al.,“Preparation of the Active Isomer of1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol, Inhibitor ofGlucocerebroside Synthetase,” J. Lipid Res., 28:565-571 (1987)).

Comparison of the two pairs of aliphatic inhibitors (bottom of Table 3)showed that the 2R,3R (D-threo) form is the primary inhibitor ofglucosyltransferase. This finding is in agreement with previousidentification of the active PDMP isomer as being the D-threoenantiomer. However, unlike the aromatic analog, BML-129 (2R,3S/2S,3R),there was a relatively small but significant activity in the case of the(erythro) 2R,3S stereoisomer. The erythro form of PDMP was found toinhibit cell proliferation of rabbit skin fibroblasts almost as well asR,R/S,S-PDMP but it did not act on the GSLs (Uemura, K. et al., “Effectof an Inhibitor of Glucosylceramide Synthesis on Cultured Rabbit SkinFibroblasts,” J. Biochem., (Tokyo) 108:525-530 (1990)). As noted withthe aromatic analogs, the pyrrolidine ring was more effective than themorpholine ring (Table 3).

Comparison of the aliphatic and corresponding aromatic inhibitors can bemade in the case of the optically active morpholine compounds PPMP andIV-206A, both of which have the R,R structure and the same fatty acid.Here it appears that the aliphatic compound is more effective (Table 3).However, in a second comparison, at lower concentrations with theinhibitors incorporated into the substrate liposomes, the degree ofinhibition was 77±0.9% with 3 μM IV-231B and 89±0.6% with 6 μM DL-threoBML-119.

Evaluations of cultured cell growth. Exposure of five different cancercell lines to inhibitors at different concentrations for 4 or 5 daysshowed that the six BML compounds most active against GlcCer synthasewere very effective growth inhibitors (Table 4). The IC₅₀ values(rounded off to one digit in the table) ranged from 0.7 to 2.6 μM.

TABLE 4 Inhibition of Tumor Cell Growth In Vitro by Various InhibitorsCELL BML- BML- TYPE BML-115 BML-118 BML-119 BML-123 BML-126 129 130MCF-7 2 2 2 2 1 3 2 H-460 2 2 1 1 1 2 3 HT-29 2 1 2 1 2 2 9L 2 2 1 2 2 22 UMSC 1 1 1 1 2 2 C-10A

FIG. 1 shows growth and survival of 9 L gliosarcoma cells grown inmedium containing different GlcCer synthase inhibitors, as describedabove. The BML compounds were used as synthesized (mixtures of DL-threoand -erythro stereoisomers) while the PDMP and PPMP were opticallyresolved R,R isomers. The concentrations shown are for the mixed racemicstereoisomers, since later work (Table 4) showed that both forms werevery similar in effectiveness. FIG. 1 illustrates the relatively weakeffectiveness of R,R-PPMP and even weaker effectiveness of R,R-PDMP. Thethree new compounds, however, are much better inhibitors of GlcCersynthase and growth. These differences in growth inhibitory powercorrelate with their effectiveness in MDCK cell homogenates as GlcCersynthase inhibitors. Some differences can be expected due to differencesin sensitivity of the synthase occurring in each cell type (thesynthases were assayed only in MDCK cells).

Growth inhibition by each of the most active BML compounds occurred inan unusually small range of concentrations (e.g., the slopes of thecytotoxic regions are unusually steep). Similar rapid drop-offs wereseen in another series of tests with 9 L cells, in which BML-119 yielded71% of the control growth with 1 μM inhibitor, but only 3% of controlgrowth with 3 μM. Growth was 93% of control growth with 2 μM BML-130 butonly 5% of controls with 3 μM inhibitor. While some clinically usefuldrugs also show a narrow range of effective concentrations, this is arelatively uncommon relationship.

When the erythro- and threo-stereoisomeric forms of BML-119 (-129 and-130) were compared, they were found to have similar effects on tumorcell growth (Table 4). This observation is similar to the results withPDMP isomers in fibroblasts cited above (Uemura, K. et al., “Effect ofan Inhibitor of Glucosylceramide Synthesis on Cultured Rabbit SkinFibroblasts,” J. Biochem., (Tokyo) 108:525-530 (1990)). Since enzymesare optically active and since stereoisomers and enantiomers of drugscan differ greatly in their effect on enzymes, it is likely that BML-129and BML-130 work on different sites of closely related metabolic steps.

FIG. 2 shows the amount of cellular protein per dish for MDCK cellscultured for 24 h in medium containing different concentrations of theseparated erythro- and threo-isomers of BML-119, as percent of theincorporation by cells in standard medium. Each point shown in FIG. 2 isthe average of values from three plates, with error bars correspondingto one standard deviation.

FIG. 3 shows [³H]thymidine incorporation into DNA of MDCK cellsincubated as in FIG. 2. The values in FIG. 3 are normalized on the basisof the protein content of the incubation dishes and compared to theincorporation by cells in standard medium.

FIGS. 2 and 3 thus provide comparison of the two stereoisomers with MDCKcells. The isomers were found to inhibit growth and DNA synthesis withsimilar effectiveness. Thus, the MDCK cells behaved like the human tumorcells with regard to IC₅₀ and the narrow range of concentrationsresulting in inhibition of protein and DNA synthesis.

Surprisingly, the aliphatic inhibitor IV-231B exerted no inhibitoryeffect on MDCK cell growth when incubated at 20 μM for 1 day or 1 μM for3 days. Tests with a longer growth period, 5 days, in 5 μM inhibitoralso showed no slowing of growth. The dishes of control cells, whichcontained BSA as the only additive to the medium, contained 3.31±0.19 mgof protein, while the IV-231B/BSA treated cells contained 3.30±0.04 mg.

Lipid changes induced in the cells. Examination by TLC of thealkali-stable MDCK lipids after a 24 h incubation disclosed that BML-130was more effective than BML-129 in lowering GlcCer levels, as expectedfrom its greater effectiveness in vitro as a glucosyltransferaseinhibitor. The level of GlcCer, estimated visually, was greatly loweredby 0.3 μM BML-130 or 0.5 μM BML-129. The levels of the other lipidsvisible on the plate (mainly sphingomyelin (SM), cholesterol, and fattyacids) were changed little or not at all. BML-129 and the GlcCersynthase inhibitor, BML-130, were readily detected by TLC at the variouslevels used, showing that they were taken up by the cells during theincubation period at dose-dependent rates. Lactosylceramide overlappedthe inhibitor bands with solvent D but was well separated with solventE, which brought the inhibitors well above lactosylceramide.

Ceramide accumulation was similar for both stereoisomers (data notshown). An unexpected finding is that noticeable ceramide accumulationappeared only at inhibitor concentrations that were more than enough tobring GlcCer levels to a very low point (e.g., at 2 or 4 μM). Thechanges in ceramide concentration were quantitated in a separateexperiment by the diglyceride kinase method, which allows one to alsodetermine diacylglycerol (DAG) concentration (Preiss, J. E. et al.,“Quantitative Measurement of SN-1,2-Diacylglycerols Present inPlatelets, Hepatocytes, and Ras- and Sis-Transformed Normal Rat KidneyCells,” J. Biol. Chem., 261:8597-8600 (1986)). The results (Table 5) aresimilar to the visually estimated ones: at 0.4 μM BML-129 or -130 therewas little effect on ceramide content but at 4 μM inhibitor, asubstantial increase was observed. (While the duplicate protein contentsper incubation dish were somewhat erratic in the high-dose dishes, inwhich growth was slow, the changes were nevertheless large and clear.)Accumulation of ceramide had previously been observed with PDMP, at asomewhat higher level of inhibitor in the medium (Shayman, J. A. et al.,“Modulation of Renal Epithelial Cell Growth by Glucosylceramide:Association with Protein Kinase C, Sphingosine, and Diacylglyceride,” J.Biol. Chem., 266:22968-22974 (1991)). From the data for cellular proteinper incubation dish, it can be seen that there was no growth inhibitionat the 0.4 μM level with either compound but substantial inhibition atthe 4 μM level, especially with the glucosyltransferase inhibitor,BML-130. This finding is similar to the ones made in longer incubationswith human cancer cells.

TABLE 5 Effects of BML-129 and -130 on MDCK Cell Growth and the Contentof Ceramide and Diacylglycerol Protein Ceramide Diglyceride GrowthMedium μg/dish nmol/mg protein Controls 490 1.04 4.52 560 0.96 5.61 0.4μm BML-129 500 1.29 5.51 538 0.99 5.13 0.4 μm BML-130 544 0.94 4.73 5380.87 5.65   4 μm BML-129 396 3.57 9.30 311 3.78 9.68   4 μm BML-130 1605.41 11.9 268 3.34 8.71

In a separate study of ceramide levels in MDCK cells, BML-130 at variousconcentrations was incubated with the cells for 24 h. The ceramideconcentration, measured by TLC densitometry, was 1.0 nmol/mg protein at0.5 μM, 1.1 ar 1 μM, 1.5 at 2 μM, and 3.3 at 4 μM. The results withBML-129 were virtually identical.

It is interesting that the accumulation of ceramide paralleled anaccumulation of diacylglycerol (DAG), as observed before with PDMP(Shayman, J. A. et al., “Modulation of Renal Epithelial Cell Growth byGlucosylceramide: Association with Protein Kinase C, Sphingosine, andDiacylglyceride,” J. Biol. Chem., 266:22968-22974 (1991)). DAG isordinarily considered to be an activator of protein kinase C and thus agrowth stimulator, but the low level of GlcCer in the inhibited cellsmay counteract the stimulatory effect. Ceramide reacts with lecithin toform SM and DAG, so it is possible that the increased level of thelatter reflects enhanced synthesis of the phosphosphingolipid ratherthan an elevated attack on lecithin by phospholipase D.Arabinofuranosylcytosine (ara-C), an antitumor agent, also produces anelevation in the DAG and ceramide of HL-60 cells (Strum, J. C. et al.,“1-β-D-Arabinofuranosylcytosine Stimulates Ceramide and DiglycerideFormation in HL-60 Cells,” J. Biol. Chem., 269:15493-15497 (1994)).

TLC of MDCK cells grown in the presence of 0.02 to 1 μM IV-231B for 3days showed that the inhibitor indeed penetrated the cells and thatthere was a great depletion of GlcCer, but no ceramide accumulation. Thedepletion of GlcCer was evident even at the 0.1 μM level and virtuallyno GlcCer was visible at the 1 μM level; however, the more polar GSLswere not affected as strongly. After incubation for 5 days in 5 μMinhibitor, all the GSLs were virtually undetectable. The ceramideconcentrations in the control and depleted cells were very similar:13.5±1.4 vs 13.9±0.2 μg/mg protein.

The lack of ceramide accumulation in cells exposed to the aliphaticinhibitors was examined further to see if it might be due todifferential actions of the different inhibitors on additional enzymesinvolving ceramide metabolism. For example, IV-231B might block ceramidesynthase and thus prevent accumulation despite the inability of thecells to utilize ceramide for GlcCer synthesis. However, assay ofceramide synthase in homogenized cells showed it was not significantlyaffected by 5 μM inhibitors (Table 6). There did appear to be moderateinhibition at the 50 μM level with PDMP and the aliphatic inhibitor.

TABLE 6 Effect of Inhibitors on Acid and Neutral Ceramidases andCeramide Synthase of MDCK Cells Enzyme Activity (% of control)Ceramidase Ceramidase Ceramide Inhibitor Tested pH 4.5 pH 7.4 SynthaseD-threo-PDMP, 5 μM  97 ± 4 116 ± 19  99 ± 5 D-threo-PDMP, 50 μM 133 ±13^(a) 105 ± 11  66 ± 9^(a) BML-129, 5 μM 108 ± 8 100 ± 0  97 ± 0BML-129, 50 μM 171 ± 26^(a)  99 ± 2 102 ± 1 BML-130, 5 μm 107 ± 11 100 ±15 108 ± 10 BML-130, 50 μm 160 ± 21^(a) 100 ± 15 106 ± 29 IV-231B, 5 μm106 ± 3 116 ± 20  90 ± 8 IV-231B, 50 μm 113 ± 8 112 ± 3  71 ± 18^(a)^(a)Notable differences.

Assay of the two kinds of ceramidase (Table 6) showed that there was noeffect of either the aliphatic or aromatic inhibitors at the 5 μM level,at which point cell growth is completely stopped in the case of thepyrrolidino compounds. At the 50 μM level, however, the acid enzyme wasstimulated markedly by the aromatic inhibitors, particularly the twostereoisomeric forms of the pyrrolidino compound.

Sphingomyelin synthase was unaffected by PDMP or the aliphatic inhibitorbut BML-129 and -130 produced appreciable inhibition at 50 μM (54% and61%, respectively) (Table 7).

TABLE 7 Effect of Inhibitors on Acid and Neutral Sphingomyelinases andSphingomyelin Synthase Enzyme Activity (% of control) InhibitorSphingomy- Sphingomyelinase Sphingomyelinase Tested elinase pH 4.5 pH7.1 Synthase^(a) D-threo-PDMP, 102 ± 3 121 ± 13 5 μM D-threo-PDMP, 100 ±3 108 ± 8 50 μM BML-129, 5 μM 108 ± 4 105 ± 11 84 ± 27 BML-129, 50 μM 97 ± 3 142 ± 11^(b) 46 ± 11^(b) BML-130, 5 μM 109 ± 1 110 ± 7 87 ± 14BML-130, 50 μM 114 ± 2 152 ± 14 39 ± 18^(b) IV-231B, 5 μM 101 ± 7 131 ±3^(b) IV-231B, 50 μM 112 ± 11 120 ± 3^(b) ^(a)Data for PDMP and IV-231Bare not shown here as they were tested in other experiments; no effectwas seen. ^(b)Notable differences.

Neutral sphingomyelinase (SMase) was distinctly stimulated by thealiphatic inhibitor, IV-231B, even at 5 μM (Table 7). From this onewould expect that the inhibitor would produce accumulation of ceramide,yet it did not. The two pyrrolidino compounds produced appreciablestimulation at the 50 μM level. No significant effects were obtainedwith acid SMase.

Discussion

The present invention shows that the nature and size of the tertiaryamine on ceramide-like compounds exerts a strong influence on GlcCersynthase inhibition, a 5-membered ring being most active. It also showsthat the phenyl ring used previously to simulate the trans-alkenyl chaincorresponding to that of sphingosine could, with benefit, be replacedwith the natural alkenyl chain.

Findings with the most active GlcCer synthase inhibitors in growth testscompare favorably with evaluations of some clinically usefulchemotherapeutic agents on three of the tumor cell lines in the sameDrug Evaluation Core Laboratory. The IC₅₀ values were 0.2 to 6 μM forcisplatin, 0.02 to 44 μM for carboplatin, 0.03 to 0.2 μM formethotrexate, 0.07 to 0.2 μM for fluorouracil, and 0.1 to 1 μM foretoposide. Unlike these agents, the compounds of the present inventionyielded rather similar effects with all the cell types, including MDCKcells, and thus have wider potential chemotherapeutic utility. Thisuniformity of action is consistent with the idea that GSLs play a wideand consistent role in cell growth and differentiation.

An important observation from the MDCK cell study is that stronginhibition of cell growth and DNA synthesis occurred only at the sameconcentrations of aromatic inhibitor that produced marked ceramideaccumulation. This observation supports the assertion that ceramideinhibits growth and enhances differentiation or cell death (Bielawska,A. et al., “Modulation of Cell Growth and Differentiation by Ceramide,”FEBS Letters, 307:211-214 (1992)). It also agrees with previous workwith octanoyl sphingosine, a short chain ceramide that produced greatlyelevated levels of natural ceramide and slowed growth (Abe, A. et al.,“Metabolic Effects of Short-Chain Ceramide and Glucosylceramide onSphingolipids and Protein Kinase C,” Eur. J. Biochem., 210:765-773(1992)). It is also in agreement with a finding that some synthetic,nonionic ceramide-like compounds did not inhibit GlcCer synthase eventhough they behave like ceramide in blocking growth (Bielawska, A. etal., “Ceramide-Mediated Biology. Determination of Structural andStereospecific Requirements Through the Use of N-Acyl-PhenylaminoalcoholAnalogs,” J. Biol. Chem, 267:18493-18497 (1992)). Compounds testedincluded 20 μM D-erythro-N-myristoyl-2-amino-1-phenyl-1-propanol, itsL-enantiomer, the four stereoisomers of N-acetylsphinganine, andN-acetylsphingosine. Furthermore, the lack of growth inhibition andceramide accumulation in cells treated with the aliphatic inhibitorIV-231B is also consistent with the correlation between ceramide leveland growth rate.

The accumulation of ceramide that occurred at higher levels of GlcCersynthase inhibitors could be attributed not only to blockage of ceramideutilization, but also to blockage of SM synthesis or ceramide hydrolase.This possibility is especially relevant to the R,S-, S,R-, andS,S-isomers, which seem to exert effects on sphingolipids withoutstrongly inhibiting GlcCer synthesis. The tests with both theDL-erythro-pyrrolidino inhibitor (BML-129) and the DL-threo-pyrrolidinoinhibitor (BML-130), at a level producing strong growth inhibition,showed that neither material at a low concentration inhibited theenzymes tested in vitro (Tables 6 and 7) but they did cause growthinhibition as well as accumulation of ceramide. PDMP, at relatively highconcentrations (50 μM), was found to inhibit SM synthase in growing CHOcells (Rosenwald, A. G. et al., “Effects of a Sphingolipid SynthesisInhibitor on Membrane Transport Through the Secretory Pathway,”Biochemistry, 31:3581-3590 (1992)). In the test with MDCK homogenates,it did not inhibit this synthase, in agreement with the finding thatlabeled palmitate incorporation into SM was stimulated by PDMP (Shayman,J. A. et al., “Modulation of Renal Epithelial Cell Growth byGlucosylceramide: Association with Protein Kinase C, Sphingosine, andDiacylglyceride,” J. Biol. Chem., 266:22968-22974 (1991)).

Retinoic acid is a growth inhibitor of interest in cancer chemotherapyand a possible adjunct in the use of the inhibitors of the presentinvention. It has been found to elevate ceramide and DAG levels (Kalen,A. et al., “Elevated Ceramide Levels in GH4C1 Cells Treated withRetinoic Acid,” Biochim. Biophys. Acta, 1125:90-96 (1992)) and possiblylower lecithin content (Tang, W. et al., “Phorbol Ester Inhibits13-Cis-Retinoic Acid-induced Hydrolysis of Phosphatidylinositol4,5-Bisphosphate in Cultured Murine Keratinocytes: a Possible NegativeFeedback Via Protein Kinase C-Activation,” Cell Bioch. Funct., 9:183-191(1991)).

D-threo-PDMP was found to be rather active in delaying tumor cell growthor in producing complete cures in mice (Inokuchi, J. et al., “AntitumorActivity in Mice of an Inhibitor of Glycosphingolipid Biosynthesis,”Cancer Lett, 38:23-30 (1987)) but high doses were needed. From the datain FIG. 1, the inhibitors of the present invention are approximately 30times as active, so the dosage levels are typical of clinically usefuldrugs. The need to use high doses with PDMP was attributed to rapidinactivation by cytochrome P450 (Shukla, A. et al., “Metabolism ofD-[³H]PDMP, an Inhibitor of Glucosylceramide Synthesis, and theSynergistic Action of an Inhibitor of Microsomal Monooxygenase,” J.Lipid Res., 32:713-722 (1991)). Cytochrome P450 can be readily blockedby various nontoxic drugs such as cimetidine, therefore high levels ofthe compounds of the present invention can be maintained.

SPECIFIC EXAMPLE 2

A series of inhibitors based on substitutions in the phenyl ring of P4were synthesized and studied. It was found that the potency of theinhibitors in blocking GlcCer synthase was mainly dependent uponhydrophobic and electronic properties of the substituent. Surprisingly,a linear relationship was found between log [IC₅₀] and hydrophobicparameter (π)+electronic parameter (δ). This correlation suggested thatelectron donating and hydrophilic characters of the substituent enhancethe potency as an inhibitor. This observation resulted in the synthesisof novel compounds that are more active in blocking glucosylceramideformation. Two compounds, dioxy D-t-P4 compounds,D-t-3′,4′-ethylenedioxy-P4 and D-t-4′-hydroxy-P4, were observed to besignificantly more potent than other tested inhibitors. In particular,at 11.3 nM D-t-3′,4′-ethylenedioxy-P4, 80% of glucosylceramide in MDCKcell was depleted without any ceramide accumulation and cell growthinhibition. The potency of D-t-3′,4′-ethylenedioxy-P4 appears to be notonly regulated by hydrophobic and electronic properties but also bystearic properties of the substituents on the phenyl group.

Materials and Methods

Materials. The acetophenones and amines were from Aldrich Chemical Co.,St. Louis, Mo., Lancaster Synthesis Inc., Windham, N.H. and MaybridgeChemical Co., Cornwall, UK. Silica gel for column chromatography (70-230mesh ASTM) and Silica gel thin layer chromatography plates werepurchased from Merck Co. The reagents and their sources were:non-hydroxy fatty acid ceramide from bovine brain and delipidated bovineserum albumin (BSA) from Sigma; dioleoyphosphatidylcholine from Avanti;DL-dithiothreitol from Calbiochem; 1-[³H]-glucose uridine diphosphatefrom NEN. Octanoylsphingosine, glucosylceramide and sodium sulfatidewere prepared as previously described. Abe, A. et al., Eur. J.Biochemistry, 210:765-773 (1992).

General synthesis of inhibitors. The aromatic inhibitors weresynthesized by the Mannich reaction from 2-N-acylaminoacetophenone,paraformaldehyde, and pyrrolidine, and then the reduction from sodiumborohydride as described before. Inokuchi, J. et al., J. Lipid. Res.,28:565-571 (1987); Abe, A. et al., J. Lipid. Res., 36:611-621 (1995).The reaction produces a mixture of four isomers, due to the presence oftwo asymmetric centers. For these syntheses in which phenyl-substitutedstarting materials were used, the chloro, methoxy, methylenedioxy,methyl groups in the acetophenone structure were brominated andconverted to the primary amine. Bromation of the methoxyacetophenone,dimethyoxyacetophenone, 3′,4′-(methylenedioxy)acetophenone wereperformed in chloroform at room temperature and recrystallized fromethyl acetate and hexane.

Synthesis of1-(4′-hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol. Thesynthesis of1-(4′-hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol isdescribed in detail in FIG. 8. This synthesis differs from those of theother compounds because of the need for the placement of a protectinggroup on the free hydroxyl (step 1) and its subsequent removal (step 7).All other syntheses employ a similar synthetic scheme (steps 2 to 6).

4′-Benzyloxyacetophenone formation (step 1) 4′-Hydroxyacetophenone(13.62 g, 100 mmol), benzylbromide (17.1 g, 100 mmol), and cesiumcarbonate (35.83 g, 100 mmol) were added to tetrahydrofuran at roomtemperature and stirred overnight. The product was concentrated todryness and recrystallized from ether and hexane to yield 15 g of4′-benzyloxyacetophenone which appeared as a white powder. An R_(f) of0.42 was observed when resolved by thin layer chromatography usingmethylene chloride. ¹H nmr (δ, ppm, CDCl₃), 7.94 (2H, δ, 8.8 Hz,O—Ar—C(O)), 7.42 (5H, m, Ar′CH₂O—), 7.01 (2H, δ, 8.8 Hz, O—Ar—C(O)),5.14 (2H, s, Ar′CH₂O—), 2.56 (3H, S, CH₃).

Bromination of 4′-benzyloxyacetophenone (step 2) Bromine (80 mmol) wasadded dropwise over 5 min to a stirred solution of4′-benzyloxyacetophenone (70 mmol) in 40 ml chloroform. This mixture wasstirred for an additional 5 min and quenched with saturated sodiumbicarbonate in water until the pH reached 7. The organic layers werecombined, dried over MgSO₄, and concentrated to dryness. The crudemixture was purified over a silica gel column and eluted with methylenechloride to yield 2-bromo-4′-benyloxyacetophenone. An R_(f) of 0.62 wasobserved when resolved by thin layer chromatography using methylenechloride. ¹H nmr (δ, ppm, CDCl₃), 7.97 (2H, δ, 9.2 Hz, O—Ar—(O)), 7.43(5H, m, Ar′CH₂O—), 7.04 (2H, δ, 9.0 Hz, O—Ar—C(O)), 5.15 (2H, s,Ar′CH₂O—), 4.40 (2H, s, CH₂Br).

2-Amino-4′-benzyloxyacetophenone HCl formation (step 3)Hexamethylenetetramine (methenamine, 3.8 g, 23 mmol) was added to astirred solution of 2-bromine-4′-benyloxyacetophenone (6.8 g, 23 mmol)in 100 ml chloroform. After 4 h the crystalline adduct was filtered andwashed with chloroform. The product was dried and heated with 150 mlmethanol and 8 ml of concentrated HCl in an oil bath at 85° C. for 3 h.Upon cooling the precipitated hydrochloride salt (2.5 g) was removed byfiltration. The filtrate was left at −20° C. overnight and additionalproduct (2.1 g) was isolated. The yield was 4.6 g (82.6%). [M⁺H]⁺: 242for C₁₅H₁₆NO₂. ¹H nmr (δ, ppm, CDCl₃), 8.38 (2H, bs, NH₂), 7.97 (2H, δ,8.8 Hz, O—Ar—C(O)), 7.41 (5H, m, Ar′CH2O—), 7.15 (2H, δ, 8.6 Hz,OArC(O)), 5.23 (2H, s, Ar′CH₂O), 4.49 (2H, s, CH₂NH₂).

2-Palmitoylamino-4′-benyloxyacetophenone formation (step 4) Sodiumacetate (50% in water, 29 ml) was added in three portions to a stirredsolution of 2-amino-4′-benzyloxyacetophenone HCl (4.6 g, 17 mmol) andtetrahydrofuran (200 ml). Palmitoyl chloride (19 mmol) intetrahydrofuran (25 ml) was added dropwise over 20 min yielding a darkbrown solution. The mixture was stirred overnight at room temperature.The aqueous fraction was removed by use of a separatory funnel andchloroform/methanol (2/1, 150 ml) was added to the organic layer whichwas then washed with water (50 ml). The yellow aqueous layer wasextracted once with chloroform (50 ml). The organic solutions were thenpooled and rotoevaporated until near dryness. The residue wasredissolved in chloroform (100 ml) and crystallized by the addition ofhexane (400 ml). The flask was cooled to 4° C. for 2 h. The crystalswere filtered and washed with cold hexane and dried in a fume hoodovernight. The product yield was 3.79 g (8 mmol). An R_(f) of 0.21 wasobserved when resolved by thin layer chromatography using methylenechloride. [M⁺H]⁺: 479 for C₃₁H₄₅NO₃. ¹H mm (δ, ppm, CDCl₃), 7.96 (2H, δ,8.8 Hz, O—Ar—C(O)), 7.40 (5H, m, Ar′CH₂O—), 7.03 (2H, δ, 8.8 Hz,O—Ar—C(O)), 6.57 (1H, bs, NH₂), 5.14 (2H, s, Ar′CH₂O—), 4.71 (2H, s,C(O)CH₂NHC(O)), 2.29 (2H, t, 7.4 Hz, C(O)CH₂(CH₂)₁₃CH₃), 1.67 (2H, m,C(O)CH₂(CH₂)₁₃CH₃), 0.87 (3H, t, 6.7 Hz, C(O)CH₂(CH₂)₁₃ CH₃).

1-(4′-Benzyloxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanolformation (steps 5 and 6) 2-Palmitoylamino-4′-benzyloxyacetophenone(3.79 g, 8.0 nmol), paraformaldehyde (0.25 g, 2.7 mmol), pyrrolidine(0.96 ml, 11.4 mmol) and ethanol (70 ml) were stirred under nitrogen.Concentrated HCl (0.26 ml) was added through the condensor and themixture was heated to reflux for 16 h. The resultant brown solution wascooled on ice and then sodium borohydride (1.3 g, 34 mmol) was added inthree portions. The mixture was stirred at room temperature overnight,and the product was dried in a solvent evaporator. The residue wasredissolved in dichloromethane (130 ml) and hydrolyzed with 3N HCl(pH˜4). The aqueous layer was extracted twice with dichloromethane (50ml). The organic layers were pooled and washed twice with water (30 ml),twice with saturated sodium chloride (30 ml), and dried over anhydrousmagnesium sulfate. The dichloromethane solution was rotoevaporated to asemisolid and purified by use of a silica rotor using a solventconsisting of 10% methanol in dichloromethane. This yielded a mixture ofDL-threo- and DL-erythro enantiomers (2.53 g, 4.2 mmol). An R_(f) of0.43 for the erythro diastereomers and 0.36 for the threo diastereomerswas observed when resolved by thin layer chromatography usingmethanol:methylene chloride (1:9). [M⁺H]⁺: 565 for C₃₆H₅₆N₂O₃.

1-(4′-Hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol formation(step 7): A suspension of 20% Pd/C (40 mg) in acetic acid (15 ml) wasstirred at room temperature under a hydrogen balloon for 15 min.1-(4′-Benzyloxy)phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (420mg, 0.74 mmol) was added and the solution was stirred overnight. Thesuspension was filtered through a glass frit, and the filter was rinsedwith acetic acid:methylene chloride (1:1, 5 ml). The filtrate wasconcentrated in vacuo and crystallized to yield a pale yellow semisolid(190 mg, 0.4 mmol). An R_(f) of 0.21 was observed when resolved by thinlayer chromatography using methanol:methylene chloride (1:9). [M⁺H]⁺:475 for C₂₉H₅₀N₂O₃. ¹H nmr (δ, ppm, CDCl₃), 7.13 (4H, m, ArCHOH—), 7.14(1H, δ, 6.9 Hz, —NH—), 5.03 (1H, δ, 3.3 Hz, CHOH—), 4.43 (1H, m,c-(CH₂CH₂)₂NCH₂CH), 3.76 (2H, m, c-(CH₂CH₂)₂N—), 3.51 (1H, m,c-(CH₂CH₂)₂NCH₂—), 3.29 (1H, m, c-(CH₂CH₂)₂NCH₂—), 2.97 (3H, m,c-(CH₂CH₂)₂N— and ArC(OH)H—), 2.08 (6H, m, —C(O)CH₂(CH₂)₁₃CH₃ andc-(CH₂CH₂)₂N—, 1.40 (2H, m, C(O)CH₂CH₂(CH₂)₁₂CH₃), 1.25 (2H, m,—C(O)CH₂CH₂(CH₂)₁₂CH₃), 0.87 (3H, t, 6.7 Hz, C(O)CH₂(CH₂)₁₃CH₃).

Synthesis ofD-threo-1-(3′,4′-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol

2-Amino-3′,4′-(ethylenedioxy)acetophenone HCl: Hexamethylenetetramine(methenamine, 5.4 g, 0.039 mol) was added to a stirred solution ofphenacylbromide (10.0 g, 0.039 mol) in 200 ml chloroform. After 2 h, thecrystalline adduct was filtered and washed with chloroform. The productwas then dried and heated with methanol (200 ml) and concentrated HCl(14 ml) in an oil bath at 85° C. for 2 h. On cooling, the precipitatedammonium chloride was removed by filtration and the filtrate was left ina freezer overnight. After filtration the crystallized phenacylamine HClwas washed with cold isopropanol and then with ether. The yield of thisproduct was ˜7.1 g (81%).

2-Palmitoylamino-3′,4′-(ethylenedioxy)acetophenone: AminoacetophenoneHCl (7.1 g, 31 mmol) and tetrahydrofuran (300 ml) were placed in a 1liter three-neck round bottom flask with a large stir bar. Sodiumacetate (50% in water, 31 ml) was added in three portions to thissuspension. Palmitoyl chloride (31 ml, 10% excess, 0.036 mol) intetrahydrofuran. (25 ml) was then added dropwise over 20 min to yield adark brown solution. This mixture was then stirred for an additional 2 hat room temperature. The resultant mixture was poured into a separatoryfunnel to remove the aqueous solution. Chloroform/methanol (2/1, 150 ml)was then added to the organic layer and washed with water (50 ml). Theyellow aqueous layer was extracted once with chloroform (50 ml). Theorganic solutions were pooled and rotoevaportated until almost dry. Theresidue was redissolved in chloroform (100 ml) and crystallized by theaddition of hexane (400 ml). The flask was then cooled to 4° C. for 2 h.The crystals were filtered and washed with cold hexane until they werealmost white and then dried in a fume hood overnight. The yield of theproduct was 27 mmol (1.6 g).

D-threo-1-(3′,4′-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol:Almitoylaminoacetophenone (11.6 g, 0.027 mol), paraformaldehyde (0.81 g,0.009 mol), pyrrolidine (3.6 ml, 0.042 mmol) and ethanol (250 ml) wereadded to a 500 ml round flask under nitrogen flow. Concentrated HCl (0.8ml) was added to this mixture through the reflux condenser and themixture was refluxed for 16 h. The brown solution was cooled in anice-bath. Sodium borohydride (2.28 g, 0.06 mol) was added in threeportions. This mixture was stirred at room temperature for 3 h and thenrotoevaporated. The residue was dissolved in 130 ml of dichloromethaneand the borate complex hydrolyzed with HCl (3N) until the pH wasapproximately 4. The aqueous layer was extracted twice with 50 mldichloromethane. The organic layers were pooled and washed twice withH₂O (30 ml), saturated NaCl (30 ml) and dried over anhydrous MgSO₄. Thedichloromethane solution was rotoevaporated to a viscous oil which waspurified by use of a Chromatotron with a solvent consisting of 10%methanol in dichloromethane to obtain a mixture of DL-threo and erythroenantiomers (2.24 g, 0.004 mol).

Resolution of inhibitor enantiomers. High performance liquidchromatography (HPLC) resolution of the enantiomers of DL-threo andDL-erythro are performed using a preparative HPLC column (Chirex 3014:[(S)-val-(R)-1-a-naphtyl)ethylamine, 20×250 mm: Phenomenex], eluted withhexane-1,2-dichloroethane-ethanol-trifluroacetic acid 64:30:5.74:0.26,at a flow rate of 8 ml/min. The column eluent was monitored at 254 nm inboth the preparative and analytical modes. Isolated products werereinjected until pure by analytical HPLC analysis, determined using ananalytical Chirex 3014 column (4.6×250 mm) and the above solvent mixtureat flow rate of 1 ml/min.

Glycosylceramide synthase activity. The enzyme activity was measured bythe method previously described in Skukla, G. et al., Biochim. Biophys.Acta, 1083:101-108 (1991). MDCK cell homogenate (120 μg of protein) wasincubated with uridinediphosphate [³H]glucose (100,000 cpm) andliposomes consisting of 85 μg octanoylsphingosine, 570 μgdioleoyphosphatidylcholine and 100 μg sodium sulfatide in 200 μl ofreaction mixture and kept for 1 h at 37° C. P4 and P4 derivativesdissolved in dimethyl sulfoxide were dispersed into the reaction mixtureafter adding liposomes. The final concentration of dimethyl sulfoxidewas kept 1% under which the enzyme activity was not at all inhibited.

Cell culture and lipid extraction. One half million of MDCK cells wereseeded into 10 cm style dish containing 8 ml serum free DMEMsupplemented medium. Shayman, J. A. et al., J. Biol. Chem.,265:12135-12138 (1990). After 24 h the medium was replaced with 8 ml ofthe medium containing 0, 11.8, 118 or 1180 nM D-t-P4,D-t-3′,4′-ethylenedioxy-P4 or D-4′-hydroxy)-P4. The GlcCer synthaseinhibitors were added into the medium as a one to one complex withdelipidated BSA. Abe, A. et al., J. Lipid. Res., 36:611-621 (1995); Abe,A. et al., Biochim. Biophys. Acta, 1299:331-341 (1996). The cells wereincubated for 24 h or 48 h with the inhibitors. After the incubation,the cells were washed twice with 8 ml of cold PBS and fixed with 2 ml ofcold methanol. The fixed cells were scraped and transferred to a glasstube. Another one ml of methanol was used to recover the remaining cellsin the dish.

Three ml of chloroform was added to the tube and briefly sonicated usinga water bath type sonicator. After centrifugation at 800 g for 5 min,the supernatant was transferred into another glass tube. The residueswere reextracted with chloroform/methanol (1/1). After thecentrifugation, the resultant supernatant was combined with the firstone. The residues were air-dried and kept for protein analysis. Adding0.9% NaCl to the supernatant combined, the ratio ofchloroform/methanol/aqueous was adjusted to 1/1/1. After centrifugation800 g for 5 min, the upper layer was discarded. Methanol/water (1/1)with the same amount of volume of the lower layer was used to wash. Theresultant lower layer was transferred into a small glass tube and drieddown under a stream of nitrogen gas. A part of the lipid was used forlipid phosphate determination. Ames, B. N., Methods Enzymol., 8:115-118(1966). The remainder was analyzed using HPTLC (Merck).

Results

Synthesis of P4 and P4 derivatives. The preparation of P4 derivativesutilized the Mannich reaction from 2-N-acylaminoacetophenone,paraformaldehyde, and pyrrolidine, and then the reduction ofDL-pyrrodino ketone from sodium borohydride. In most cases, no isolationof DL-pyrrodino ketones were performed to maintain solubility. Theoverall yields of the DL-threo and DL-erythro syntheses were ˜10-30%.These derivatives were purified by the either silica gel column orrotors with solvent 5-12% methanol in dichloromethane to optimize theseparation from the chiral column. To obtain the best separation, eachinjection contains no more than 150 mg, and fractions were pooled toobtain sufficient quantity of isomer of D-threo for further biologicalcharacterization.

Resolution of PDMP homologues by chiral chromatography. The structuresof the parent compound, D-threo-P4 and the phenyl-substituted homologuesincluding the new dioxy-substituted and 4′-hydroxy-P4 homologues areshown in FIG. 9. Initially the effect of each P4 isomer separated bychiral chromatography on GlcCer synthase activity was determined (FIG.10). Four peaks were observed for the chiral separation of P4. Peaks 1and 2 represented the erythro diastereomers and 3 and 4 represented thethreo diastereomers as determined by a sequential separation of the P4mixture by reverse phase chromatography followed by the chiralseparation. The enzyme activity was specifically inhibited by the fourthpeak, the D-threo isomer (FIG. 4A). This specificity for the D-threoenantiomer was consistent with the previous results observed in PDMP andPDMP homologues (2-4). The IC₅₀ of D-threo-P4 was 0.5 mM for GlcCersynthase activity measured in the MDCK cell homogenates.

Effects of P4 and P4 Derivatives with a Single Substituent of PhenylGroup on GlcCer Synthase Activity. The effect of each P4 isomer onGlcCer synthase activity was analyzed. The reaction was carried out inthe absence or presence of 0.1, 1.0 or 10 μM P4 (FIG. 4A) orp-methoxy-P4 (FIG. 4B). As shown in FIG. 4A, the enzyme activity wasspecifically inhibited by D-threo isomer. In FIG. 4A, the symbols aredenoted as follows: D-threo (∘), D-erythro (□), L-threo and (•),L-erythro (Δ). This specificity is consistent with previous resultsobserved in PDMP and PDMP homologs. Inokuchi, J. et al., J. Lipid. Res.28:565-571 (1987); Abe, A. et al., J. Lipid. Res. 36:611-621 (1995). TheIC₅₀ of D-t-P4 was 500 nM.

As set forth herein, the addition of a p-methoxy group to DL-t-P4 wasfound to enhance the effect of the inhibitor on the enzyme activity.Abe, A. et al., J. Lipid. Res., 36:611-621 (1995). As shown in FIG. 4B,it was confirmed that the enzyme activity was potently inhibited byD-threo-p-methoxy-P4 whose IC₅₀ was 200 nM. In FIG. 4B, □ denotes amixture of D-erythro and L-threo isomers contaminated with a smallamount of the D-threo isomer. Chiral chromatography of the fourp-methoxy-P4 enantiomers failed to completely resolve to baseline eachenantiomer (FIG. 10). A slight inhibition of the enzyme activity byp-methyoxy-P4 in a combined D-erythro and L-threo mixture (peaks 2 and3, FIG. 10) was observed; this was due to contamination of the D-threoisomer (peak 4, FIG. 10) into these fractions.

A series of D-t-P4 derivatives containing a single substituent on thephenyl group were investigated. As shown in Table 8, the potency of thederivatives as inhibitors were inferior to that of D-t-P4 orp-methoxy-D-t-P4. In many drugs, the influence of an aromaticsubstituent on the biological activity has been known and predicted.Hogberg, T. et al., Theoretical and experimental methods in drug designapplied on antipsychotic dopamine antagonists. Larsen, P. K., andBundgaard, H., “Textbook of Drug Design and Development,” pp. 55-91(1991). Generally IC₅₀ is described as the following equation:log(1/IC ₅₀)=a(hydrophobic parameter(π)+b(electronicparameter(σ)+c(stearic parameter)+d(other descriptor)+ewhere a, b, c, d and e are the regression coefficients. Hogberg, T. etal., Theoretical and experimental methods in drug design applied onantipsychotic dopamine antagonists. Larsen, P. K., and Bundgaard, H.,“Textbook of Drug Design and Development,” pp. 55-91 (1991).

The hydrophobic effect, π, is described by the equation π=log P_(x)−logP_(H) where P_(X) is the partition coefficient of the substitutedderivative and P_(H) is that of the parent compound, measured as thedistribution between octanol and water.

The electronic substituent parameter, σ, was originally developed byHammett (Hammett, L. P., In Physical Organic Chemistry, McGraw-Hill, NewYork (1940)) and is expressed as σ=log K_(X)−log K_(H), where K_(X) andK_(H) are the ionization constants for a para or meta substitutedderivative and benzoic acid respectively. Positive σ values representelectron withdrawing properties and negative a values represent electrondonating properties.

The potency of D-threo-P4 and P4 derivatives as an inhibitor is mainlydependent upon two factors, hydrophobic and electronic properties, of asubstituent of phenyl group (Table 8). Surprisingly, a linearrelationship was observed between log (IC₅₀) and π+σ (FIG. 5). Thesefindings suggest that the more negative the value of π+σ, the morepotent is D-threo-P4 derivatives made as GlcCer synthase inhibitor.

The data in Table 8 indicate that the potency of D-t-P4 and P4derivatives as an inhibitor is mainly dependent upon two properties,hydrophobic and electronic properties, of a substituent of the phenylgroup. Surprisingly, a linear relationship was observed betweenlog(IC₅₀) and π+σ (FIG. 5). These findings suggest that the morenegative the value of π+σ, the more potent the D-t-P4 derivative as aGlcCer synthase inhibitor.

TABLE 8 D-threo-P4 derivative σ + π* IC₅₀ (μM)** p-methoxy −0.29 0.2 P-40.00 0.5 m-methoxy-P4 0.10 0.6 p-methyl-P4 0.39 2.3 p-chloro-P4 0.94 7.2*These values were estimated from the Table in Hogberg, T. et al.,Theoretical and experimental methods in drug design applied onantipsychotic dopamine antagonists. Larsen, P. K., and Bundgaard, H.,“Textbook of Drug Design and Development,” pp. 55–91 (1991),for methoxy,σ_(m) 0.12, σ_(p) = −0.27, π = −0.02; hydro, σ = 0, π = 0; methyl, σ_(p)= −0.17, π = 0.56; chloro, σ_(p) = 0.23, π = 0.71. **These values werederived from FIGS. 4A and 4B. For other compounds the same analyticalapproach as shown in FIGS. 4A and 4B was carried out to obtain the IC₅₀.

The p-hydroxy-substituted homologue was a significantly better GlcCersynthase inhibitor. The strong association between π+σ and GlcCersynthase inhibition suggested that a still more potent inhibitor couldbe produced by increasing the electron donating and decreasing thelipophilic properties of the phenyl group substituent. A predictablynegative π+σ value would be observed for the p-hydroxy homologue. Thiscompound was synthesized and the D-threo enantiomer isolated by chiralchromatography. An IC₅₀ of 90 nM for GlcCer synthase inhibition wasobserved (FIG. 11), suggesting that the p-hydroxy homologue was twice asactive as the p-methoxy compound. Moreover, the linear relationshipbetween the log (IC₅₀) and π+σ was preserved (open circle, FIG. 4).

Effects of 3′,4′-dioxy-D-threo-P4 Derivatives on GlcCer SynthaseActivity. The result in FIG. 5 suggested that an electron donating andhydrophilic substituent of phenyl group makes the GlcCer synthaseinhibitor potent. To attain further improvement of the inhibitor,another series of P4 derivatives with methylenedioxy, ethylenedioxy andtrimethyldioxy substitutions on the phenyl group were designed (FIG. 9).

As shown in FIG. 6, the enzyme activity was markedly inhibited byD-t-3′,4′-ethylenedioxy-P4 whose IC₅₀ was 100 nM. In FIG. 6, □ denotesD-t-3′,4′-methylenedioxy-P4, ∘ denotes D-t-3′,4′-ethylenedioxy-P4, Δdenotes D-t-3′,4′-trimethylenedioxy-P4 and • denotesD-t-3′,4′-dimethyoxy-P4. One the other hand, the IC₅₀ s forD-t-3′,4′-methylenedioxy-P4 and D-t-3′,4′-trimethylenedioxy-P4 wereabout 500 and 600 nM, respectively. These results suggest that thepotency of D-t-3′,4′-ethylenedioxy-P4 is not only regulated byhydrophobic and electronic properties but also by other factors, mostlikely stearic properties, induced from the dioxy ring on the phenylgroup.

Interestingly, D-t-3′,4′-dimethoxy-P4 was inferior to these dioxyderivatives, even to D-t-P4 or m- or D-t-p-methoxy-P4, as an inhibitor(FIG. 6). As the parameters, σ_(m), σ_(p) and π, for methoxy substituentare 0.12, −0.27 and −0.02, respectively (Hogberg, T. et al., Theoreticaland experimental methods in drug design applied on antipsychoticdopamine antagonists. Larsen, P. K., and Bundgaard, H., “Textbook ofDrug Design and Development,” pp. 55-91 (1991)), the value of π+σ ofD-t-dimethoxy P4 is presumed to be negative. Therefore, the dimethoxy-P4is thought to deviate quite far from the correlation as observed in FIG.5. There may be a repulsion between two methoxy groups in thedimethoxy-P4 molecule that induces a stearic effect that was negligiblein mono substituent D-t-P4 derivatives studied in FIG. 5. GlcCersynthase is thought to possess a domain that interacts with D-t-PDMP andPDMP homologs and that modulates the enzyme activity. Inokuchi, J. etal., J. Lipid. Res., 28:565-571 (1987); Abe, A. et al., Biochim.Biophys. Acta, 1299:331-341 (1996). The stearic effect generated by anadditional methoxy group may affect the interaction between the enzymeand the inhibitor. As a result, the potency as an inhibitor is markedlychanged.

Distinguishing Between Inhibition of GlcCer Synthase and1-O-acylceramide Synthase Inhibition. Prior studies on PDMP and relatedhomologues revealed that both the threo and erythro diastereomers werecapable of increasing cell ceramide and inhibiting cell growth in spiteof the observation that only the D-threo enantiomers blocked GlcCersynthase. An alternative pathway for ceramide metabolism wassubsequently identified, the acylation of ceramide at the 1-hydroxylposition, which was blocked by both threo and erythro diastereomers ofPDMP. The specificities of D-threo-P4, D-threo-3′,4′-ethylenedioxy-P4,and D-threo-(4′-hydroxy)-P4 for GlcCer synthase were studied by assayingthe transacylase. Although there was an ca. 100 fold difference inactivity between D-threo-3′,4′-ethylenedioxy-P4,D-threo-(4′-hydroxy)-P4, and D-threo-P4 (IC₅₀ 0.1 mM versus 10 mM) ininhibiting GlcCer synthase, the D-threo enantiomers of all threecompounds demonstrated comparable activity in blocking 1-O-acylceramidesynthase (FIG. 12).

In order to determine whether inhibition of 1-O-acylceramide synthasewas the basis for inhibitor mediated ceramide accumulation, the ceramideand diradylglycerol levels of MDCK cells treated D-threo-P4,D-threo-3′,4′-ethylenedioxy-P4, and D-threo-(4′-hydroxy)-P4 weremeasured (Table 9). MDCK cells (5×10.sup.5) were seeded into a 10 cmdish and incubated for 24 h. Following the incubation, the cells weretreated for 24 or 48 h with or without P4 or the phenyl substitutehomologues. Both ceramide and diradylglycerol contents were determinedby the method of Preis, J. et al., J. Biol. Chem., 261:8597-8600 (1986).GlcCer content was measured densitometrically by a video camera and useof NIH image 1.49. Significant increases in both ceramide anddiradylglycerol occurred only in cells treated with inhibitorconcentrations in excess of 1 mM. This was approximately 30-fold lowerthan the concentration required for inhibition of the 1-O-acylceramidesynthase assayed in the cellular homogenates. This disparity inconcentration effects most likely reflects the ability of the morepotent homologues to accumulate within intact cells. Abe, A. et al.,Biochim. Biophys. Acta, 1299:331-341 (1996).

TABLE 9 GlcCer, ceramide and diradylglycerol content of MDCK cellstreated with D-threo-P4, D-threo-3′,4′-ethylenedioxy-P4, andD-threo-(4′-hydroxy)-P4 Ceramide Diradylglycerol (pmol/nmol (pmol/nmolCondition phospholipids) phospholipids) Control 24 h 4.53 ± 0.12 24.2 ±2.36 48 h 6.68 ± 0.49 32.3 ± 3.11 D-threo-P4 11.3 nM 24 h  5.33 ± 0.41*24.1 ± 1.66 48 h  5.68 ± 0.27* 29.6 ± 0.73 113 nM 24 h 4.64 ± 0.38 26.6± 1.56 48 h 7.08 ± 0.29 33.0 ± 2.63 1130 nM 24 h 5.10 ± 0.35 27.1 ± 0.6748 h 9.74 ± 0.53 38.8 ± 1.11 D-threo-4′-hydroxy- 11.3 nM P4 24 h 4.29 ±0.71  30.9 ± 2.01* 48 h 6.70 ± 0.29  38.4 ± 1.44* 113 nM 24 h 5.09 ±0.95  31.5 ± 3.84* 48 h 7.47 ± 0.29  41.5 ± 0.66* 1130 nM 24 h 7.38 ±0.13  38.5 ± 3.84* 48 h  13.4 ± 1.03*  47.2 ± 2.51* D-threo-3′,4′- 11.3nM ethylenedioxy-P4 24 h 5.24 22.0 5.04 24.7 113 nM 24 h 5.21 32.5 5.2141.6 1130 nM 24 h 9.64 32.5 13.0 41.6 *Denotes p < 0.05 by the Student ttest. For the D-threo-(ethylenedioxy)-P4 only two determinations weremade.

Effects of D-threo-P4, D-threo-4′-hydroxy-P4 andD-threo-3,4′-ethylenedioxy-P4 on GlcCer Synthesis and Cell Growth. Toconfirm the cellular specificity of D-threo-3′,4′-ethylenedioxy-P4 andD-threo-(4′-hydroxy)-P4 as compared to D-threo-P4, MDCK cells weretreated with different concentrations of the inhibitors. The totalprotein amount in each sample was determined by the BCA method. InGlcCer analysis, lipid samples and standard lipids were applied to thesame HPTLC plate pre-treated with borate and developed in a solventconsisting of C/M/W (63/24/4). The level of GlcCer was estimated from astandard curve obtained using a computerized image scanner. The valueswere normalized on the basis of the phospholipid content. The resultsare shown in FIG. 7, wherein each bar is the average values from threedishes, with error bars corresponding to one standard deviation. In thecontrol, the total protein and GlcCer were 414±47.4 μg/dish and24.3±1.97 ng/nmol phosphate, respectively.

Approximately 66 and 78% of the GlcCer was lost from the cells treatedby 11.3 nM D-threo-4′-hydroxy-P4 and D-threo-3′,4′-ethylenedioxy-P4respectively (FIGS. 7, 14 and 15). By contrast, only 27 percentdepletion of GlcCer occurred in cells exposed to D-threo-P4 (FIG. 13). Alow level of GlcCer persisted in the cells treated with 113 or 1130 nMof either compound. This may be due to the contribution, by degradation,of more highly glycosylated sphingolipids or the existence of anotherGlcCer synthase that is insensitive to the inhibitor.

On the other hand, there was little difference in the total proteincontent between untreated and treated cells with 11.3 or 113 nMD-threo-4′-hydroxy-P4 and D-threo-3′,4′-ethylenedioxy-P4 (FIGS. 14 and15). A significant decrease in total protein was observed in the cellstreated with 1130 nM of either P4 homologue. In addition, the level ofceramide in the cells treated with 1130 nMD-threo-3′,4′-ethylenedioxy-P4 and D-threo-(4′-hydroxy)-P4 was two timeshigher than that measured in the untreated cells (Table 9). There was nochange in ceramide or diradylglycerol levels in cells treated with 11.3nM or 113 nM concentrations of either compound. Similar patterns forGlcCer levels and protein content were observed at 48 h incubations.

The phospholipid content was unaffected at the lower concentrations ofeither D-threo-3′,4′-ethylenedioxy-P4 or D-threo-(4′-hydroxy)-P4. Theratios of cell protein to cellular phospholipid phosphate (mgprotein/nmol phosphate) were 4.94±0.30, 5.05±0.21, 4.84±0.90, and3.97±0.29 for 0, 11.3, 113, and 1130 nM D-threo-3′,4′-ethylenedioxy-P4respectively, and 4.52±0.39, 4.35±0.10, and 3.68±0.99 for 11.3, 113, and1130 nM D-threo-4′-hydroxy-P4 suggesting that the changes in GlcCercontent were truly related to inhibition of GlcCer synthase activity.These results strongly indicate that the inhibitorsD-threo-4′-hydroxy-P4 and D-threo-3′,4′-ethylenedioxy-P4, are able topotently and specifically inhibit GlcCer synthesis in intact cells atlow nanomolar concentrations without any inhibition of cell growth.

SPECIFIC EXAMPLE 3

Compositions within the scope of invention include those comprising acompound of the present invention in an effective amount to achieve anintended purpose. Determination of an effective amount and intendedpurpose is within the skill of the art. Preferred dosages are dependentfor example, on the severity of the disease and the individual patient'sresponse to the treatment.

As used herein, the term “pharmaceutically acceptable salts” is intendedto mean salts of the compounds of the present invention withpharmaceutically acceptable acids, e.g., inorganic acids such assulfuric, hydrochloric, phosphoric, etc. or organic acids such asacetic.

Pharmaceutically acceptable compositions of the present invention mayalso include suitable carriers comprising excipients and auxiliarieswhich facilitate processing of the active compounds into preparationswhich may be used pharmaceutically. Such preparations can beadministered orally (e.g., tablets, dragees and capsules), rectally(e.g. suppositories), as well as administration by injection.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, e.g., using theconventional mixing, granulating, dragee-making, dissolving orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding a resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars, e.g.,lactose or sucrose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, e.g., tricalcium diphosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, e.g., maizestarch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose and/or polyvinylpyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, e.g., silica,talc, stearic acid or salts thereof, such as magnesium stearate orcalcium stearate, and/or polyethylene glycol. Dragee cores are providedwith suitable coatings which, if desired, are resistant to gastricjuices. For this purpose, concentrated sugar solutions may be used,which may optionally contain gum arabic, talc, polyvinylpyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvent or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used. Dyestuffs or pigments may be added to thetablets or dragee coatings, e.g., for identification or in order tocharacterize different combinations of active compound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules may contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycols. In addition, stabilizers maybe used.

Possible pharmaceutical preparations which can be used rectally include,e.g., suppositories, which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases are, e.g.,natural or synthetic triglycerides, paraffin hydrocarbons, polyethyleneglycols or higher alkanols. It is also possible to use gelatin rectalcapsules which consist of a combination of the active compounds with abase. Possible base materials include, e.g., liquid triglycerides,polyethylene glycols or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, e.g.,water-soluble salts. In addition, suspension of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, such as sesame oil,or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension such as sodium carboxymethylcellulose,sorbitol and/or dextran. Optionally, the suspension may also containstabilizers.

Alternatively, the active compounds of the present invention may beadministered in the form of liposomes, pharmaceutical compositionswherein the active compound is contained either dispersed or variouslypresent in corpuscles consisting of aqueous concentrate layers adherentto hydrophobic lipidic layer. The active compound may be present both inthe aqueous layer and in the lipidic layer or in the non-homogeneoussystem generally known as a lipophilic suspension.

The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the accompanyingdrawings, that various changes, modifications and variations can be madetherein without departing from the spirit and scope of the invention.

All publications cited herein are expressly incorporated by reference.

1. A pharmaceutical composition comprising a compound represented by thefollowing structural formula:

or a stereoisomer, a pharmaceutically acceptable salt or a mixturethereof; and a pharmaceutically acceptable carrier or an excipient,wherein: R¹ is a phenyl, a substituted phenyl group, a branchedaliphatic group, or a 7-15 carbons long alkyl chain or a 7-15 carbonslong alkenyl chain with a double bond next to the kernel; R² is an alkylgroup 6, 7, or 8 carbons long; and R³ is a pyrrolidine, azetidine orpiperidine, in which the nitrogen atom is attached to the kernel.
 2. Thepharmaceutical composition of claim 1, wherein R¹ is a phenyl groupsubstituted with a functional group selected from the group consistingof a p-methoxy, hydroxyl, methylenedioxy, ethylenedioxy,trimethylenedioxy and cyclohexyl.
 3. A pharmaceutical compositioncomprising a compound represented by the structural formula:

or a stereoisomer, a pharmaceutically acceptable salt or a mixturethereof; and a pharmaceutically acceptable carrier or an excipient,wherein: R¹ is a substituted or unsubstituted phenyl group, a branchedaliphatic group, or a 7-15 carbons long alkyl chain or a 7-15 carbonslong alkenyl chain with a double bond next to the kernel; R² is an alkylgroup 6, 7, or 8 carbons long; and R³ is pyrrolidine, in which thenitrogen atom is attached to the kernel.
 4. The pharmaceuticalcomposition of claim 3, wherein R¹ is a phenyl group substituted with afunctional group selected from the group consisting of a p-methoxy,hydroxyl, methylenedioxy, ethylenedioxy, trimethylenedioxy andcyclohexyl.